CN104093890B - The composition produced for the enrichment of target nucleotide sequence and efficient library and method - Google Patents

The composition produced for the enrichment of target nucleotide sequence and efficient library and method Download PDF

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CN104093890B
CN104093890B CN201380006942.4A CN201380006942A CN104093890B CN 104093890 B CN104093890 B CN 104093890B CN 201380006942 A CN201380006942 A CN 201380006942A CN 104093890 B CN104093890 B CN 104093890B
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sequence
adapter
nucleic acid
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CN104093890A (en
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道格·阿莫莱赛
克里斯·阿穆尔
努里斯·库恩
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Nugen Technologies Inc
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Abstract

The invention provides and carry out the method for high-efficiency nucleic acid library generation, composition and test kit for the target nucleotide sequence enrichment in nucleic acid samples with for new-generation sequencing (NGS).Especially, method provided herein, composition and test kit can be used for producing from the nucleic acid samples containing compound DNA and catch interested amplification ready, the region specific with chain of target specificity.

Description

The composition produced for the enrichment of target nucleotide sequence and efficient library and method
cross reference
This application claims the U.S. Provisional Application the 61/591st submitted on January 26th, 2012, the rights and interests of No. 241, this provisional application is incorporated to herein by reference.
background of invention
Along with developing rapidly of new-generation sequencing (NGS) technology and platform, genome sequencing becomes further feasible.Researchist is forced to produce increasing data volume to obtain the more deep understanding to variation and biology trend, and is forced to produce data to avoid multiple intercellular equalization in tissue from less sample size.
Although genome sequencing cost is declining and the flux of NGS platform constantly increases, interested genome area is being selected to carry out checking order and analyze often actual and meet cost benefit.Target enrichment be genomic dna order-checking in normally used strategy, wherein before order-checking from DNA sample selectivity target acquisition genome area.The target enrichment paid close attention to is a kind of important means, in research (such as, to the research based on colony of disease marker or the SNP) field particularly making genome sequencing cost be difficult to accept checking order to a large amount of sample.Similarly, the improvement obtained makes to prepare DNA library by the nucleic acid from less amount cell becomes possibility, but these improve the restriction being limited to ligation efficiency.
Developed many target enriching methods, these methods are different from each other in sensitivity, specificity, circulation ratio, homogeneity, cost and ease for use.Current normally used target enriching method can be divided into 3 primary categories, and each classification respectively has the Pros and Cons of its uniqueness: the 1) method of PCR-based; 2) catching by hybridization, i.e. hybrid capture on array or in solution; And 3) catching by cyclisation, namely based on the method for molecular inversion probes.
The method of PCR-based uses highly parallel pcr amplification, and each target sequence wherein in sample has the sequence specific primers of corresponding a pair uniqueness.Use while a large amount of primer pair because high-caliber non-specific amplification and primer-primer interact and make multiplex PCR unrealistic.Nearest exploitation, droplet round pcr (people such as Tewhey, 2009) that wherein each amplified reaction is separated into single droplet physically can eliminate multiplex PCR and non-specific amplification and primer-primer and to interact relevant restriction.But, the method of droplet PCR and other PCR-based improved needs special instrument or platform, and its flux is restricted, and when the target area of enriched, equally need a large amount of single primer pairs with conventional multiplex PCR, therefore make target enrichment become expensive.
Hybrid capture method is the selective cross of the oligonucleotide designed based on targeted genome region and user.This hybridization can be the hybridization (array is caught) with the oligonucleotide be fixed in high-density or low density microarray, or can be and the solution phase hybridization (catching in solution) that can be fixed to the oligonucleotide that solid surface is modified as the part (biological example element) on pearl subsequently.In order to effectively catch, hybrid capture needs compound pond (pool) and the long hybridization cycle (normally 48 hours) of expensive long oligonucleotide.For hybrid capture on array, also need expensive instrument and hardware.Because the efficiency of hybridization is relatively low, so need a large amount of input DNA.
Method based on molecular inversion probes (MIP) depends on and builds a large amount of linear oligonucleotide probes of strand, and the common linker that this probe is target specific sequence by flank forms.After annealing with target sequence, by be polymerized and connection fills probe gap regions, thus produce circularizing probes.Then discharge circularizing probes, and use the primer for common linker region to increase.One of main drawback based on the target enrichment of MIP is that it relatively low catches homogeneity, means the variability that existence is larger in the sequential covering of whole target region.The same with PCR and hybrid capture, the method based on MIP needs target specificity oligonucleotide that in a large number may be expensive.
Allow low cost to not needing to use special instrument, the selectivity target enriching method of improvement that high-throughout target genome area is caught exists demand.In addition, also demand is existed to the generation of high-efficiency nucleic acid library.Method of the present invention as herein described meets these demands.
Summary of the invention
In one aspect, disclosed herein is the method for enrich target target nucleic acid sequence in the sample comprising nucleic acid, the method comprises: (a) fragmented nucleic acids, thus produces nucleic acid fragment; B first adapter is attached to 5 ' end of each nucleic acid fragment by (); C () makes one or more oligonucleotide and nucleic acid fragment anneal, thus each making in described one or more oligonucleotide comprises and 3 ' part of the target nucleic acid array complementation be present in one or more described nucleic acid fragment and comprise 5 ' part of the second adapter sequence; D () with one or more oligonucleotide described in polymerase extension, thus produces and one or morely has the first adapter at the first end and have the oligonucleotide extension products of the second adapter at the second end; And (e) use with the first primer of the first adapter complementation and with one or more oligonucleotide extension products described in the second primer amplification of the second adapter complementary, to be enriched in the nucleic acid fragment that each end comprises the first adapter and the second adapter sequence.In one embodiment, to the additional step that described one or more oligonucleotide extension products checks order after the method is included in amplification further.In one embodiment, target nucleotide sequence comprises genomic dna, RNA or cDNA.In one embodiment, target nucleotide sequence comprises genomic dna.In one embodiment, target nucleotide sequence comprises cDNA.In one embodiment, the method makes nucleic acid fragment sex change before being included in step c further, thus produces the single-chain nucleic acid fragment at 5 ' end with the first adapter sequence.In one embodiment, the first adapter can be common for each nucleic acid fragment.In one embodiment, the second adapter sequence can be common for described one or more oligonucleotide.In one embodiment, the first adapter and the second adapter sequence can be different from each other.In one embodiment, the first adapter and/or the second adapter sequence comprise bar code sequence further.In one embodiment, step b can be undertaken by connection.In one embodiment, the method is included in further after the first adapter connects and carries out breach reparation to produce the additional step with the nucleic acid fragment of spacer end.In one embodiment, the composition comprising the target nucleotide sequence of enrichment is produced by method disclosed herein.In one embodiment, this polysaccharase can be archaeal dna polymerase.
On the other hand, disclosed herein is the method for enrich target target nucleic acid sequence in the sample comprising nucleic acid, the method comprises: (a) fragmented nucleic acids, thus produces nucleic acid fragment, b first adapter is attached to this nucleic acid fragment by (), wherein the first adapter comprises the partial duplex with short chain and long-chain, wherein 3 ' end of the short chain of this partial duplex adapter comprises blocking groups, and 5 ' end of the long-chain of this partial duplex adapter comprises restriction for nucleic acid modifying enzyme and/or cleavage site, c () makes this nucleic acid fragment sex change, thus produce single-chain nucleic acid fragment, d () makes one or more oligonucleotide and this single-chain nucleic acid fragment anneal, thus each making in described one or more oligonucleotide comprises the sequence with the target nucleic acid array complementation be present in one or more described single-chain nucleic acid fragment, with oligonucleotide one or more described in polymerase extension to produce one or more double-strandednucleic acid mixture, this mixture comprises target nucleic acid sequence and complementary sequence thereof, have for the double-strand restriction of nucleic acid modifying enzyme and/or the first end of cleavage site and second end of 3 ' overhang with the short chain comprising the first adapter, e () cuts the restriction of this double-strand and/or cleavage site with nucleic acid modifying enzyme, thus produce cleavage site, f second adapter is connected to this cleavage site by (), wherein the second adapter comprises the duplex with two chains, g () makes the sex change of described one or more double-strandednucleic acid mixture, thus producing one or more single-chain nucleic acid fragment, this fragment comprises the short chain of target nucleic acid sequence, the chain from the second adapter in the first end and the first adapter in the second end, and (h) use comprises with the first primer of the sequence of the chain complementation from the second adapter and comprises the described one or more single-chain nucleic acid fragments comprising described one or more target nucleic acid sequence with the second primer amplification of the sequence of the short chain complementation of the first adapter, thus one or more target nucleic acid sequence described in enrichment.In one embodiment, to the additional step that the one or more single-chain nucleic acid fragments from step h check order after the method is included in amplification further.In one embodiment, target nucleotide sequence comprises genomic dna, RNA or cDNA.In one embodiment, target nucleotide sequence comprises genomic dna.In one embodiment, target nucleotide sequence comprises cDNA.In one embodiment, the first adapter and the second adapter can be common for each nucleic acid fragment.In one embodiment, the first adapter and the second adapter can be different from each other.In one embodiment, the first adapter and/or the second adapter comprise bar code sequence further.In one embodiment, from the restriction of the double-strand for nucleic acid modifying enzyme of step e and/or cleavage site comprise the partial duplex long-chain of the first adapter 5 ' end and by one or more oligonucleotide extend produce, with the sequence of 5 ' termini-complementary of the partial duplex long-chain of the first adapter.In one embodiment, this nucleic acid modifying enzyme comprises restriction enzyme.In one embodiment, step b is undertaken by connecting.In one embodiment, the composition comprising the target nucleotide sequence of enrichment is produced by method disclosed herein.In one embodiment, this polysaccharase can be archaeal dna polymerase.
In another, disclosed herein is the method for generation of nucleotide sequence library, the method comprises: (a) fragmentation comprises the sample of nucleic acid, thus produces nucleic acid fragment; B first adapter is attached to each nucleic acid fragment by (); C () makes nucleic acid fragment sex change, thus produce single-chain nucleic acid frag-ment libraries; D () makes one or more oligonucleotide and this single-chain nucleic acid fragment anneal, each in wherein said one or more oligonucleotide comprises and 3 ' part of the complementary in one or more described single-chain nucleic acid fragment and the 5 ' part comprising the second adapter sequence; E () uses one or more oligonucleotide described in polymerase extension, thus produce one or more oligonucleotide extension products, and this oligonucleotide extension products comprises the first adapter in the first end and comprises the second adapter sequence in the second end; And (f) uses one group to have one or more oligonucleotide extension products described in specific primer amplification to the first adapter and the second adapter sequence, to produce the library of the nucleic acid fragment comprising the first adapter and the second adapter sequence in each end.In one embodiment, the method is included in further after forward adapter connects and carries out breach reparation reaction to produce the additional step with the nucleic acid fragment of spacer end.In one embodiment, the method comprises the additional step checked order to one or more nucleic acid extension products of the amplification from step f further.In one embodiment, this nucleotide sequence comprises genomic dna.In one embodiment, this nucleotide sequence comprises cDNA.In one embodiment, step c can be omitted, and wherein nucleic acid fragment is double-strand.In one embodiment, 3 ' part of one or more oligonucleotide of steps d comprises stochastic sequence.In one embodiment, step b is undertaken by connecting.In one embodiment, the first adapter and the second adapter sequence can be common for each nucleic acid fragment.In one embodiment, the first adapter and the second adapter sequence can be different from each other.In one embodiment, the first adapter and/or the second adapter sequence comprise bar code sequence further.In one embodiment, method disclosed herein can produce the composition comprising nucleotide sequence library.In one embodiment, this polysaccharase can be archaeal dna polymerase.
Further, disclosed herein is for there is the first adapter and the method at the second end with the library enrich target target nucleic acid sequence of the Nucleic acid inserts of the second adapter from being included in the first end, the method comprises: (a) makes Nucleic acid inserts sex change, thus produces the library of single-chain nucleic acid Insert Fragment; B () makes one or more oligonucleotide and this single-chain nucleic acid Insert Fragment anneal, each in wherein said one or more oligonucleotide comprises and 3 ' part of the target nucleic acid array complementation be present in one or more described Nucleic acid inserts and the 5 ' part comprising the 3rd adapter sequence; C () with one or more oligonucleotide described in polymerase extension, thus produces and one or morely has the first adapter in the first end and have the oligonucleotide extension products of the 3rd adapter sequence in the second end; And (d) use with the first primer of the first adapter complementation and with one or more oligonucleotide extension products described in the second primer amplification of the 3rd adapter complementary, thus be enriched in the nucleic acid fragment that each end comprises the first adapter and the 3rd adapter sequence.In one embodiment, the method comprises the additional step checked order to one or more oligonucleotide extension products of the amplification from steps d further.In one embodiment, target nucleotide sequence comprises genomic dna.In one embodiment, target nucleotide sequence comprises cDNA.In one embodiment, step a can omit, and wherein nucleic acid fragment can be double-strand.In one embodiment, the first adapter and the second adapter can be common for each nucleic acid fragment.In one embodiment, the 3rd adapter sequence can be common for described one or more oligonucleotide.In one embodiment, the first adapter and the second adapter can be different from each other.In one embodiment, the first adapter and the second adapter can be identical.In one embodiment, the first adapter, the second adapter and/or the 3rd adapter sequence comprise bar code sequence further.In one embodiment, method disclosed herein can produce the composition of the target nucleotide sequence comprising enrichment.
quote and be incorporated to
All publications described in this specification sheets, patent and patent application are incorporated to herein, just as pointing out that each independent publication, patent or patent application are incorporated to all by reference especially and individually with identical degree all by reference.
Accompanying drawing explanation
Novel feature of the present invention is specifically set forth in the appended claims.By reference to following description of being set forth the illustrative embodiment using the principle of the invention and accompanying drawing, the better understanding to the features and advantages of the present invention will be obtained, in the accompanying drawings:
Fig. 1 shows the selectivity target enrichment using the connection of the single forward adapter of DNA fragmentation end in DNA library to carry out.Comprise common inverse adapter sequence with the sequence specific oligonucleotide of target regional annealing in its 5 ' end, and after sequence specific oligonucleotide extends, use and to forward and reverse adapter, there is specific one group of primer and carry out PCR.
Fig. 2 shows a kind of alternative connection scheme, wherein upon connection, makes DNA-fragment-adapter mixture sex change when not carrying out otch reparation and adapter fills, thus produces connection product, wherein on each Insert Fragment, there is frayed end.
Fig. 3 shows the selectivity target enrichment using the connection of partial duplex adapter to carry out.Utilize the connection that the 5 ' end (with the respective complementary sequence of the sequence specific oligonucleotide extended) having specific nucleic acid modifying enzyme cutting part duplex adapter long-chain to double-stranded DNA allows new adapter right, and increase with the primer corresponding to new adapter subsequently.
Fig. 4 shows and uses random efficient NGS library of causing to produce.The oligonucleotide of annealing with DNA fragmentation comprises common reverse adapter sequence in its 5 ' end, and after primer extension, uses to have specific one group of primer to forward and reverse adapter and carry out PCR.
detailed Description Of The Invention
General introduction
Method of the present invention can be used for the target sequence of use one group of universal primer and the multiple restriction of adapter selective enrichment from compound DNA, thus avoids the needs to multiplex PCR and multiple primer pair.Contemplate multiple target region: such as, target area can represent all known coding regions, whole exon group, the selection area in the encoding gene group region of the selected approach of representative, known packets is containing the selected genes group region of the genome mutation relevant to phenotypic alternation, specific chromosomal whole or selection area, etc.On the other hand, method of the present invention also can be used for the generation of high-efficiency nucleic acid library.
In a word, a kind of simple, low cost, the high-throughout system prepared for target enrichment and library of method establishment of the present invention.
With detailed reference to exemplary of the present invention.Although method and composition disclosed in these will be described in conjunction with these exemplary, should be appreciated that these exemplary are not intended to limit the present invention.On the contrary, the invention is intended to contain the alternatives that can be included in spirit and scope of the invention, modification and equivalents.
In one embodiment, the invention provides the method and composition for the interested particular target sequence of enrichment from the sample comprising nucleic acid.Method as herein described carrys out enriched target sequence by using traditional two chain body adapter and/or partial duplex adapter, sequence specific oligonucleotide, restriction enzyme and connection.The method allows enriched target sequence from the specific chains of template nucleic acid further, and this target sequence can use multiple amplification method to increase further.In another embodiment, the invention provides for efficiently producing the method comprising the library of interested specific nucleic acid sequence.
In one embodiment, the invention provides the method and composition for enriched target nucleotide sequence from the sample comprising nucleic acid.In one aspect, the method comprise fragmentation input sample in nucleic acid to produce nucleic acid fragment.This nucleic acid can be DNA or RNA.This nucleic acid can be strand or double-strand.This DNA can be genomic dna or cDNA or its arbitrary combination.In one embodiment, the nucleic acid inputted in sample is double-stranded DNA.In one embodiment, the fragmentation of nucleic acid realizes by method as known in the art.Fragmentation is undertaken by Physical fragmentation methods and/or enzyme catalysis fragmentation methods.Physical fragmentation methods can comprise atomization, supersound process and/or hydrodynamic force and shear.In some embodiments, fragmentation can mechanically complete, and comprises and carries out supersound process to the nucleic acid in input sample.In some embodiments, fragmentation comprises with the nucleic acid of one or more enzymes under the condition being suitable for this one or more enzyme generation double-strand nucleic acid break in process input sample.The example that can be used for the enzyme producing nucleic acid or polynucleotide passage comprises sequence-specific and the specific nuclease of non-sequence.The limiting examples of nuclease comprises DNaseI, fragmentation enzyme (Fragmentase), restriction endonuclease, its variant and combination thereof.For carrying out the commercially available acquisition of reagent (such as, from NewEnglandBiolabs) of enzymatic fragmentation reaction.Such as, there is not Mg 1+with there is Mn ++when can random ds breakage in inducing DNA with DNaseI digestion.In some embodiments, fragmentation comprises with the nucleic acid in one or more restriction endonuclease process input sample.Fragmentation can produce has 5 ' overhang, 3 ' overhang, flush end or its fragment combined.In some embodiments, such as, when fragmentation comprises one or more restriction endonucleases of use, the cutting of sample polynucleotide can produce the overhang with predictable sequence.In some embodiments, the method comprises by standard method as known in the art such as column purification or be separated the step of fragment being carried out to size selection from sepharose.
In some embodiments, the nucleic acid inputted in sample can be changed into the nucleic acid molecule of the fragmentation with one or more certain size range or the colony of polynucleotide by fragment.In some embodiments, fragment has the mean length from about 10 to about 10,000 Nucleotide.In some embodiments, fragment has the mean length from about 50 to about 2,000 Nucleotide.In some embodiments, fragment have about 100-2500,10-1000,10-800,10-500,50-500,50-250 or 50-150 Nucleotide mean length.In some embodiments, fragment has and is less than 10, the mean length of 000 Nucleotide, such as be less than 5,000 Nucleotide, be less than 2,500 Nucleotide, be less than 2,500 Nucleotide, be less than 1,000 Nucleotide, be less than 500 Nucleotide, be such as less than 400 Nucleotide, be less than 300 Nucleotide, be less than 200 Nucleotide or be less than 150 Nucleotide.
In one embodiment, the end reparation of nucleic acid fragment can be carried out after the fragmentation of nucleic acid.End reparation can comprise the generation of flush end, non-flush end (i.e. stickiness or sticky end) or single base overhang, such as adds a dA Nucleotide by the polysaccharase of shortage 3 ' exonuclease activity to the 3' end of nucleic acid fragment.End reparation can use numerous enzyme known in the art and/or method to carry out, and includes but not limited to the test kit of commercially available acquisition, as Encore tMultraLowInputNGSLibrarySystemI.In a preferred embodiment, can carry out end reparation to produce flush end to double chain DNA fragment, wherein this double chain DNA fragment comprises 5 ' phosphoric acid and 3 ' hydroxyl.In some embodiments, double chain DNA fragment can carry out flush end polishing (blunt-endpolish) (or " end reparation ") to produce the DNA fragmentation with flush end before being connected with adapter.Double-stranded segment produces flush end undertaken by using the outstanding single stranded end of strand specific DNA exonuclease such as exonuclease 1, exonuclease 7 or its combination degraded double-stranded products.Or, by using strand specific DNA endonuclease, make double chain DNA fragment flat end (bluntended) such as but not limited to mung bean endonuclease or S1 endonuclease.Or, make double-stranded products flat end by using the outstanding single stranded end of the polysaccharase containing strand exonuclease activity such as T4DNA polysaccharase or any other polysaccharase containing strand exonuclease activity or its combination degraded double-stranded products.In some cases, the polysaccharase containing strand exonuclease activity can be hatched in the reaction mixture containing or do not contain one or more dNTP.In other cases, the double-stranded segment that the combination of single-chain nucleic acid specificity exonuclease and one or more polysaccharases can be used to produce the fragmentation of the sample by comprising nucleic acid carries out flat end.When in addition other, by filling the outstanding single stranded end of double-stranded segment to make nucleic acid fragment flat end.Such as, fragment can with polysaccharase as T4DNA polysaccharase or Klenow polysaccharase or its be combined in the existence of one or more dNTP under hatch to fill the single stranded portion of double-stranded segment.Or, by using the single-stranded overhang DeR of exonuclease and/or polysaccharase and using the filling-in of one or more polysaccharases to combine under the existence of one or more dNTP to carry out flat end to double chain DNA fragment.
In some embodiments, 5 ' and/or 3 ' end nucleotide acid sequence of the nucleic acid of fragmentation did not carry out modifying or end reparation before being connected with adapter oligonucleotide of the present invention.Such as, the fragmentation undertaken by restriction enzyme can be used for producing predictable overhang, is connected subsequently with the one or more adapter oligonucleotide comprised with the overhang of the measurable overhang complementation on nucleic acid fragment.In another example, cut by enzyme and produce predictable flush end, the nucleic acid fragment of flat end can be connected with the adapter oligonucleotide comprising flush end subsequently.In some embodiments, 1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20 or more Nucleotide can be added after end reparation, such as one or more VITAMIN B4, one or more thymus pyrimidine, one or more guanine or one or more cytosine(Cyt), to produce overhang.The nucleic acid fragment with overhang can be connected, such as, in ligation with one or more adapter oligonucleotide with complementary overhangs.Such as, the polysaccharase not relying on template can be used single VITAMIN B4 to be added into 3 ' end of the DNA fragmentation that end is repaired, and be connected with one or more adapter subsequently, each adapter has thymus pyrimidine at 3 ' end.In some embodiments, adapter oligonucleotide can be connected with flush end double stranded nucleic acid fragment, and this flush end double stranded nucleic acid fragment is by extending 3 ' end with one or more oligonucleotide, carry out 5 ' phosphorylation subsequently and modified.In some cases, can containing in the suitable damping fluid of magnesium, under the existence of one or more dNTP, use polysaccharase, such as Klenow polysaccharase or the polysaccharase in this any appropriate provided, or use terminal deoxynucleotidyl transferase, carry out the extension of 3 ' end.In some embodiments, the nucleic acid fragment with flush end can be connected with the one or more adapters containing flush end.Can in the suitable damping fluid containing ATP and magnesium, T4 polynucleotide kinase be such as used to carry out the phosphorylation of 5 ' end of nucleic acid fragment.The nucleic acid molecule that optionally can process fragmentation to carry out dephosphorization acid to 5 ' end or 3 ' end, such as, by using enzyme known in the art, such as Phosphoric acid esterase.
Method for enriched target nucleotide sequence as herein described comprises further the first adapter is attached to the nucleic acid fragment produced by method as herein described.In one embodiment, the first adapter can be forward adapter.The first adapter has been carried out to adding on the nucleic acid fragment produced by methods described herein by using ligation or initiation reaction.In one embodiment, the first adapter contains connection to the additional packet on nucleic acid fragment.In one embodiment, after the end of nucleic acid fragment is repaired, the first adapter can be connected with nucleic acid fragment.In another embodiment, after nucleic acid fragment produces, when the end not carrying out nucleic acid fragment is repaired, the first adapter can be connected with nucleic acid fragment.First adapter can be the adapter of any type as known in the art, includes but not limited to traditional two chain body or double-strand adapter, and wherein adapter comprises two complementary chains.In a preferred embodiment, the first adapter can be double-stranded DNA adapter.In one embodiment, the first adapter can be the oligonucleotide with known array, therefore allows to produce and/or use sequence specific primers, to increase to any polynucleotide adding or be connected with the first adapter and/or to check order.In one embodiment, the first adapter can be traditional two chain body adapter, and wherein the first adapter comprises sequence as known in the art.In a preferred embodiment, the first adapter can be attached to the nucleic acid fragment that produced by methods described herein from multiple directions.In a preferred embodiment, method as herein described can relate to the first duplex adapter using and comprise the double-stranded DNA with known array, and this first duplex adapter is flat end and can be combined with the double stranded nucleic acid fragment produced by methods described herein from the direction of both direction.In one embodiment, the first adapter can be connected with each nucleic acid fragment, thus makes each nucleic acid fragment comprise the first identical adapter.In other words, each nucleic acid fragment comprises the first common adapter.In another embodiment, the first adapter can be added or be connected on the library of the nucleic acid fragment produced by methods described herein, thus makes each nucleic acid fragment in this nucleic acid fragment library comprise the first adapter be connected with one or two end.
In one embodiment, the first adapter can connect or be attached to 5 ' and/or 3 ' end of the nucleic acid fragment produced by methods described herein.First adapter can comprise two chains, and wherein every bar chain comprises 3 ' free hydroxyl but two chains do not comprise 5 ' free phosphoric acid.In one embodiment, 3 ' the free hydroxyl on the first adapter every bar chain can be connected with the free 5 ' phosphate on the arbitrary end being present in nucleic acid fragment of the present invention.In this embodiment, first adapter comprises connection chain and disconnected chain, thus connection chain can be connected with 5 ' phosphoric acid on the arbitrary end of nucleic acid fragment, and otch or breach may be there is between 3 ' hydroxyl on the disconnected chain and the arbitrary end of nucleic acid fragment of the first adapter.In one embodiment, this otch or breach is filled by carrying out breach reparation reaction.In one embodiment, the DNA dependent dna-polymerases with strand-displacement activity can be used to carry out breach reparation.In one embodiment, can use have weak or without the DNA dependent dna-polymerases of strand-displacement activity to carry out breach reparation.In one embodiment, the connection chain of the first adapter can be used as the template of breach reparation or filling-in.In this embodiment, breach reparation or filling-in comprise the extension using the connection chain of the first adapter as template, and cause producing the nucleic acid fragment with complementary end or end, such as, as shown in Figure 1.In one embodiment, Taq DNA polymerase can be used to carry out breach reparation.In one embodiment, breach reparation can not be carried out after the first adapter is connected with the nucleic acid fragment produced by methods described herein, such as, as shown in Figure 2.In this embodiment, nucleic acid fragment comprises only in the first adapter sequence that 5 ' end of every bar chain connects.
Connection and the optional breach reparation of the first adapter and nucleic acid fragment produce the first adapter-nucleic acid fragment mixture.In one embodiment, the first adapter-nucleic acid fragment mixture sex change can be made.Any method as known in the art can be used to realize sex change, include but not limited to physics, heat and/or chemical modification.In one embodiment, thermally denature can be used or high-temperature denaturedly realize sex change.In one embodiment, the sex change of the first adapter-nucleic acid fragment mixture creates the single-chain nucleic acid fragment only comprising the first adapter sequence in 5 ' end of nucleic acid fragment, such as, as shown in Figure 2.In another embodiment, the sex change of the first adapter-nucleic acid fragment mixture produces the single-chain nucleic acid fragment all comprising the first adapter sequence at 5 ' end of nucleic acid fragment and 3 ' end, such as, as shown in Figure 1.
In one embodiment, can make to comprise the nucleic acid fragment sex change of the first adapter sequence being attached to 5 ' end or be attached to 5 ' end and 3 ' end two ends, to produce the single-chain nucleic acid fragment comprising the first adapter sequence being attached to 5 ' end or be attached to 5 ' end and 3 ' end two ends.In one embodiment, method of the present invention as herein described can be used for producing multiple single-chain nucleic acid fragment comprising the first adapter sequence being attached to 5 ' end or be attached to 5 ' end and 3 ' end two ends.In one embodiment, following oligonucleotide can be annealed with single-chain nucleic acid fragment, this oligonucleotide comprises with the sequence of the target complementary be present in single-chain nucleic acid fragment at the first end and comprises the sequence from the second adapter at the second end, wherein this second adapter sequence not with complementary target.In one embodiment, the second adapter sequence can carry out the sequence of self-reversal adapter.In one embodiment, target nucleotide sequence can be present in one or more single-chain nucleic acid fragment.In one embodiment, target nucleotide sequence that is different or uniqueness can be present in one or more single-chain nucleic acid fragment.In one embodiment, one or more oligonucleotide can comprise the sequence with the same target complementary be present in one or more single-chain nucleic acid fragment.In this embodiment, described one or more oligonucleotide can comprise the sequence of different piece with same target sequence or regional complementarity.In one embodiment, described different zones can be adjacent one another are.In one embodiment, described different zones can be not adjacent to each other.In preferred embodiments, comprise and comprise the second identical adapter sequence further with one or more oligonucleotide of the sequence of same target target nucleic acid sequence complementation.In another embodiment, one or more oligonucleotide can comprise the sequence from different or unique target sequence complementation that may be present in one or more single-chain nucleic acid fragment.In a preferred embodiment, comprise and comprise the second identical adapter sequence further from one or more oligonucleotide of the sequence of different or unique target nucleic acid array complementation.In one embodiment, can at 3 ' end of oligonucleotide and the second adapter sequence can at 5 ' end of oligonucleotide with the sequence of target complementary.In a preferred embodiment, the second adapter sequence and target nucleotide sequence not complementary.Like this, the second adapter sequence is as afterbody.Second adapter sequence can be conventional adapter sequence.In a preferred embodiment, the second adapter sequence can be the conventional adapter sequence being different from or being different from the first adapter sequence be attached to as mentioned above in single-chain nucleic acid fragment.In one embodiment, the second adapter sequence can have known sequence, therefore allows to produce and/or use sequence specific primers, to increase to any polynucleotide adding or be connected with the second adapter and/or to check order.In an independent embodiment, oligonucleotide can be annealed with the nucleic acid fragment comprising the first adapter sequence being attached to 5 ' end or being attached to 5 ' end and 3 ' end two ends, and does not need sex change in advance.In this embodiment, oligonucleotide annealing by this oligonucleotide and comprise 5 ' end or 5 ' end and 3 ' the end two ends being attached to double stranded nucleic acid fragment the first adapter sequence double stranded nucleic acid fragment between form triple helix or triplex carries out.In this embodiment, this double-strandednucleic acid sequence comprises target sequence, and can be present in multiple comprise be attached to 5 ' end or be attached to 5 ' and 3 ' end two ends the first adapter sequence double stranded nucleic acid fragment between.Further for this embodiment, this oligonucleotide comprises the sequence with the target sequence complementation in double stranded nucleic acid fragment.In a word, the use of following oligonucleotide allows to utilize method choice as herein described to combine also nucleic acid fragment described in enrichment subsequently, and this oligonucleotide comprises and be present in the sequence of the target complementary in the nucleic acid fragment between one or more or multiple nucleic acids fragment.
After making oligonucleotide anneal as mentioned above, polymerase extension oligonucleotide can be utilized.In one embodiment, this polysaccharase can be DNA dependent dna-polymerases.In one embodiment, this DNA dependent dna-polymerases can be any DNA dependent dna-polymerases as herein described, and the extension of oligonucleotide can be undertaken by any method as known in the art.In one embodiment, comprise the second adapter sequence (wherein this second adapter sequence not with complementary target) and can to anneal with nucleic acid fragment with the oligonucleotide of the sequence comprising the target complementary existed in the nucleic acid fragment of the first adapter be attached on and/or two ends and with polymerase extension, comprising the first adapter in the first end and the oligonucleotide extension products comprising the second adapter sequence in the second end to produce.In one embodiment, nucleic acid fragment can be present among multiple nucleic acid fragment comprising the first adapter being attached to or/or two ends.In this embodiment, only oligonucleotide extension products can be produced to the nucleic acid fragment containing target sequence.
In one embodiment, the oligonucleotide extension products produced by methods described herein can experience amplified reaction.In one embodiment, this amplified reaction can be exponential, and can carry out under all temps circulation or isothermal.In one embodiment, this amplification can be polymerase chain reaction.In one embodiment, this amplified reaction can be isothermal.In one embodiment, the oligonucleotide extension products as produced by methods described herein is contained the first adapter and contain the second adapter sequence on another end on an end.In a preferred embodiment, oligonucleotide extension products can be separated with template nucleic acid fragment, has the first adapter sequence and the single stranded oligonucleotide extension products on 3 ' end with the second adapter sequence to produce on 5 ' end.Then can use to comprise and increase with the second primer pair single stranded oligonucleotide extension products of the sequence of the second adapter complementary with comprising with the first primer of the sequence of the first adapter complementation.Like this, the oligonucleotide extension products simultaneously comprising the first and second adapter sequences is only had will to obtain amplification also therefore by enrichment.In one embodiment, the first adapter and/or the second adapter sequence can comprise identifier.In one embodiment, this identifier can be bar code sequence.In one embodiment, bar code sequence can be identical or different for the first adapter and the second adapter sequence.In one embodiment, the first adapter and/or the second adapter sequence can comprise the sequence of the downstream application that can be used in such as but not limited to order-checking.In one embodiment, the first adapter and/or the second adapter sequence can comprise flow cell (flowcell) sequence, this flow cell sequence can be used for use developed by Illumina check order with sequence measurement as herein described.
In an alternative embodiment, method of the present invention can be used for the library producing nucleic acid fragment or Insert Fragment, and wherein each nucleic acid fragment comprises adapter in one or two end.In one embodiment, adapter can be present in two ends and may be different from each other.In one embodiment, adapter can be present in two ends and can comprise identical adapter sequence.Be included in generation that two ends have a library of the Nucleic acid inserts of different adapter can relate to as above for generation of comprising the first adapter sequence and comprise the method for the oligonucleotide extension products of the second adapter sequence on the first end on another end, difference is to be combined with nucleic acid fragment and extendible oligonucleotide comprises stochastic sequence.In this embodiment, oligonucleotide comprises in 3 ' part the stochastic sequence can hybridized with one or more nucleic acid fragment, and comprises the second adapter sequence further in 5 ' part.As shown in Figure 4, oligonucleotide produces along the extension of nucleic acid fragment and corresponding first adapter and comprises the second adapter at an end and comprise the one or more products with the sequence of the first adapter complementation at another end.In one embodiment, the stochastic sequence be present in oligonucleotide can be combined with one or more Nucleic acid inserts and extend thereon.In one embodiment, the one or more oligonucleotide comprising the 3 ' part containing stochastic sequence and the 5 ' part containing the second adapter sequence can be annealed with being included in the library that one or two end of each Nucleic acid inserts contains the Nucleic acid inserts of the first adapter sequence.In one embodiment, the first adapter sequence can be identical or common for each Nucleic acid inserts.In one embodiment, the second adapter sequence can be identical or common for each in one or more oligonucleotide.In one embodiment, aforesaid method can be used for the library producing Nucleic acid inserts, and wherein each Nucleic acid inserts contains the first common adapter and contain the second common adapter sequence on the second end on an end.In one embodiment, the first adapter and the second adapter sequence can be different from each other.In one embodiment, the first adapter and the second adapter sequence can comprise identical adapter sequence.In a word, the inventive method as above can be used for the efficient generation in nucleotide sequence library.
In the another alternative embodiment of the inventive method as above, the first adapter can be the double-stranded DNA adapter comprising partial duplex, and wherein two chains of this adapter can have different length, have complementary region and outburst area at 5 ' end.In this embodiment, 5 ' end of the long-chain of partial duplex adapter can comprise for the unique site of nucleic acid modifying enzyme as restriction enzyme, and this site is non-existent in the short chain of duplex adapter.In further embodiment, by replacing the 3 ' end that short chain adapter modified by 3 ' OH base, to stop polymerase extension with blocking groups such as dideoxy nucleotide (ddCMP, ddAMP, ddTMP or ddGMP).In this embodiment, the first adapter comprising partial duplex can be connected with the nucleic acid fragment produced by methods described herein.In one embodiment, after partial duplex first adapter connects, breach reparation as above reaction can be carried out.In this embodiment, partial duplex first adapter does not carry out breach reparation reaction after connecting.In a preferred embodiment, partial duplex first adapter contains free 5 ' phosphoric acid and on long-chain, contains free 3 ' hydroxyl on short chain.In this embodiment, the connection of partial duplex adapter produces double stranded nucleic acid fragment, and wherein two ends of this double stranded nucleic acid fragment comprise long-chain and the short chain of partial duplex first adapter.Double stranded section duplex the first adapter-nucleic acid fragment mixture produces by connecting.In one embodiment, double stranded section duplex the first adapter-nucleic acid fragment mixture sex change can be made, to produce the single-chain nucleic acid fragment of the long-chain containing the first adapter on the first end and the short chain containing the first adapter on the second end.In this embodiment, the first end is 5 ' end and the second end is 3 ' end.In one embodiment, first adapter can be attached on one or more nucleic acid fragments of being produced by methods described herein, to make each nucleic acid fragment comprise the first identical adapter, or, in other words, the first adapter can be common for each Nucleic acid inserts.Comprise and can anneal with this single-chain nucleic acid fragment with the oligonucleotide of the sequence of the target sequence complementation in single-chain nucleic acid fragment or primer, and use polysaccharase to extend.In one embodiment, this polysaccharase can be DNA dependent dna-polymerases.In one embodiment, this DNA dependent dna-polymerases can be any DNA dependent dna-polymerases as described herein, and the extension of oligonucleotide is undertaken by any method as known in the art.The extension of the primer of annealing with single-chain nucleic acid fragment produces and comprise the oligonucleotide extension products with the sequence of the long-chain complementation of the first adapter on an end.In one embodiment, oligonucleotide extension products keeps hybridizing with single-chain nucleic acid fragment, and making becomes double-strand for the specific restriction of nucleic acid modifying enzyme and/or cleavage site.Double stranded site can be cut subsequently with to the specific nucleic acid modifying enzyme of double-stranded restriction sites.In one embodiment, this nucleic acid modifying enzyme can be restriction enzyme.In one embodiment, this restriction enzyme can have specificity to double-stranded restriction sites.In one embodiment, the cutting of restriction site can produce flush end or non-flush end.In one embodiment, after cutting, end reparation can be carried out by any method as herein described on the end of nucleic acid fragment.The cutting of restriction and/or cleavage site produces the site that can be connected with the second adapter.The connection of the second adapter is undertaken by any method of attachment as described herein.In one embodiment, connect the double stranded nucleic acid fragment produced containing the second adapter and containing partial duplex on the second end on the first end, wherein this partial duplex comprises 3 ' overhang of the sequence containing the first adapter short chain.Any denaturation method as herein described can be used subsequently to make double stranded nucleic acid fragment sex change, on the first end, comprise the second adapter sequence and the single-chain nucleic acid fragment comprising the sequence of the first adapter short chain on the second end to produce.In one embodiment, the first end and the second end comprise 5 ' end and 3 ' end respectively.In one embodiment, after double-stranded restriction sites cutting, the second adapter can be attached on one or more nucleic acid fragment, thus make each nucleic acid fragment comprise the second identical adapter, or in other words, the second adapter can be common for each Nucleic acid inserts.Can use subsequently the second primer had to specific first primer and have specific second primer to the sequence be present in the first adapter short chain and single-chain nucleic acid fragment is increased.In one embodiment, this amplified reaction can be exponential, and can carry out under all temps circulation or isothermal.In one embodiment, this amplification can be polymerase chain reaction.In one embodiment, this amplified reaction can be isothermal.In a word, the fragment containing the second adapter and the first adapter short chain is only had will to be increased or enrichment.The enrichment of the target fragment in library is provided in the method, and not enrichment by extend comprise with the oligonucleotide of the sequence of target complementary and produce oligonucleotide extension products when, there is not the distortion (distortion) of original DNA libraries, and this enrichment and intubating length have nothing to do.Because 3 ' end of partial duplex adapter short chain be 3 ' close, allow in this way orientation or asymmetric connection.In one embodiment, comprise and comprise reverse adapter sequence further with the oligonucleotide of the sequence of the target sequence complementation in nucleic acid fragment.In this embodiment, 3 ' part of oligonucleotide can be present in the sequence of the target sequence complementation in nucleic acid fragment, and oppositely adapter sequence can be present in 5 ' part.Further for this embodiment, reverse adapter sequence can be common or the adapter sequence of routine, and can be different from or be different from the first and/or second adapter.Further for this embodiment, aforesaid method can cause generation to comprise the second adapter on an end and on another end, comprise the single-chain nucleic acid fragment of reverse adapter sequence.After this embodiment, amplification can be carried out carry out enrichment single-chain nucleic acid fragment by using the second adapter had to specific first primer and have specific second primer to the 3rd adapter sequence.
Method of the present invention can be applicable to any enrichment from library to target nucleotide sequence further, this library is included in the nucleic acid fragment that one or two end is attached with the sample of adapter sequence, wherein this library produces as follows: the connection using adapter as described herein or adapter sequence and one or two end, or by not relying on the method for connection, such as Nextra, the i.e. a kind of method that drives of swivel base group (transposome).In one embodiment, this nucleic acid can be DNA, such as genomic dna or cDNA.In one embodiment, this nucleic acid can be double-strand.The enrichment of target nucleic acid sequence can use the method for target enrichment as herein described to realize.In one embodiment, make Nucleic acid inserts sex change to produce the library of single-chain nucleic acid Insert Fragment from comprising containing the method being attached with the library enrich target target nucleic acid sequence of the Nucleic acid inserts of adapter at one or two end.In one embodiment, each Nucleic acid inserts can comprise the first adapter sequence and in opposite ends, comprise the second adapter sequence on an end.In one embodiment, the first adapter and the second adapter can be different from each other.In one embodiment, the first adapter and the second adapter can comprise identical adapter sequence.In one embodiment, each Nucleic acid inserts can comprise the first adapter sequence and in opposite ends, comprise the second adapter sequence on an end, produces on an end, comprise the first adapter sequence and the library comprising the single-chain nucleic acid Insert Fragment of the second adapter sequence in opposite ends to make sex change.Any method as herein described can be used to realize sex change.Further for above-mentioned embodiment, one or more oligonucleotide can be annealed with single-chain nucleic acid Insert Fragment.In one embodiment, each in one or more oligonucleotide comprises and 3 ' part of the target nucleic acid array complementation be present in one or more Nucleic acid inserts and the 5 ' part comprising the 3rd adapter sequence.In one embodiment, the 3rd adapter sequence is different from any one in the first adapter and the second adapter or two.One or more oligonucleotide can extend with polysaccharase (i.e. archaeal dna polymerase), thus generation has the first or second adapter in the first end and has one or more oligonucleotide extension products of the 3rd adapter sequence at the second end.In one embodiment, the first end comprises 5 ' end and the second end comprises 3 ' end.Can use may with the first primer of the first or second adapter complementation and may with the second primer of the 3rd adapter complementary to one or more oligonucleotide extension products that increases, to be enriched in the nucleic acid fragment that each end comprises the first or second adapter and the 3rd adapter sequence.In one embodiment, the first and second adapters can be common for each Nucleic acid inserts in library.In one embodiment, the 3rd adapter sequence can be common for each in one or more oligonucleotide.In a word, target enriching method as above can be used for producing composition, and said composition comprises the Nucleic acid inserts library for the enrichment from the non-enriched library that the Nucleic acid inserts being connected to one or two end by adapter forms of any target sequence.
Fig. 1 illustrates the schematic diagram of the preferred embodiment of the methods described herein for enrich target target sequence.In a word, Fig. 1 describe from nucleic acid fragment library or multiple nucleic acid fragment be separated or enrichment comprise the nucleic acid fragment of target nucleic acid sequence or the method for Insert Fragment.The method of Fig. 1 comprises the linking library producing nucleic acid fragment or Insert Fragment, wherein each fragment of this linking library or Insert Fragment comprise common forward adapter and the fragment different from forward adapter or the reverse adapter of Insert Fragment specificity, enrichment can comprise nucleic acid fragment or the Insert Fragment of target nucleic acid sequence with the abatement PCR making to use the primer for common forward adapter and the primer for reverse adapter to carry out.The input of method shown in Fig. 1 is the DNA of fragmentation.The DNA of fragmentation is double-strand and comprises DNA fragmentation library or multiple DNA fragmentation.In one embodiment, DNA fragmentation can from compound DNA, as double-stranded DNA, genomic dna or the DNA from more than an organic mixing.In one embodiment, DNA fragmentation can be controlled oneself and be changed into the RNA of cDNA by the first chain synthesis reaction, this conversion use well known in the art, for producing any method of cDNA from RNA template, can include but not limited to by RNA and primer (i.e. random primer) combined, and use RNA dependent dna-polymerases reverse transcription RNA template.In one embodiment, DNA fragmentation can be controlled oneself and be used any method well known in the art to be changed into the RNA of double-strand cDNA by the first and second chain synthesis reactions.Realize the fragmentation of DNA to produce DNA fragmentation by any method for nucleic acid fragment described herein, the method can include but not limited to (i.e. the supersound process) of physics and/or (i.e. restriction enzyme treatment) fragmentation reaction of chemistry.
As shown in Figure 1, single forward adapter is connected with DNA fragmentation.In one embodiment, single forward adapter can comprise known sequence.In one embodiment, single forward adapter can be common adapter.In one embodiment, DNA fragmentation can experience end reparation as described herein reaction to produce flush end.In this embodiment, single forward adapter also can comprise flush end, and the connection between single forward adapter and DNA fragmentation is undertaken by flush end connection as described herein.By using enzyme (i.e. T4DNA ligase enzyme) to promote to be connected with method as known in the art, include but not limited to the test kit of commercially available acquisition, as Encore tMultraLowInputNGSLibrarySystem.In FIG, forward adapter can comprise the chain (connection chain) be connected with the free 5 ' phosphoric acid on DNA fragmentation 5 ' end and the chain (disconnected chain) be not connected with DNA fragmentation 3 ' end.In one embodiment, ligation can cause producing otch or breach between the disconnected chain and 3 ' end of DNA fragmentation of single forward adapter.In this embodiment, repair or fill otch or breach by breach reparation or filling-in, wherein can use polysaccharase (preferred DNA dependent dna-polymerases, such as Taq DNA polymerase) 3 ' end of extended DNA fragment, wherein the connection chain of forward adapter can be used as template.In this embodiment, breach reparation produces the DNA fragmentation with spacer end.As described in Figure 1, make the DNA fragmentation sex change with spacer end, to produce the sex change library comprising the Single-stranded DNA fragments with spacer end.Any method as known in the art can be used to realize sex change, and the method can include but not limited to thermally denature and/or chemical modification.
As shown in Figure 1, customization (custom) oligonucleotide with reverse adapter afterbody and the Single-stranded DNA fragments with spacer end are annealed.In one embodiment, the customization oligonucleotide with reverse adapter afterbody can comprise 3 ' part and 5 ' part, this 3 ' part comprises the sequence with the target complementary in one of Single-stranded DNA fragments, this 5 ' part comprise not with the reverse adapter sequence of the Single-stranded DNA fragments complementation in sex change library.In one embodiment, reverse adapter sequence can be known sequence.In a preferred embodiment, as described herein, reverse adapter sequence can be different from single forward adapter.In one embodiment, can multiple customization oligonucleotide with reverse adapter afterbody be added in sex change library, wherein said multiple customization oligonucleotide with reverse adapter afterbody comprises 3 ' part and 5 ' part, this 3 ' part comprises the sequence with the target complementary in one or more Single-stranded DNA fragments in sex change library, this 5 ' part comprise not with the reverse adapter sequence of the Single-stranded DNA fragments complementation in sex change library.In one embodiment, multiple each that have in the customization oligonucleotide of reverse adapter afterbody in, reverse adapter afterbody comprises identical reverse adapter sequence, and wherein oppositely adapter sequence be different from forward adapter sequence.In another one embodiment, for multiple each that have in the customization oligonucleotide of reverse adapter afterbody, reverse adapter afterbody comprises different reverse adapter sequences, and wherein each different reverse adapter sequence is different from forward adapter sequence.In one embodiment, 3 ' the part with the customization oligonucleotide of reverse adapter afterbody can be specific sequence, wherein customize oligonucleotide and comprise the sequence with target complementary, and provide a kind of means using the one or more target sequence of method target of the present invention enrichment.In another embodiment, 3 ' the part with the customization oligonucleotide of reverse adapter afterbody can be the random sequence produced, this sequence can be hybridized with the stochastic sequence of the frag-ment libraries on one or two end with adapter sequence, thus provides the means utilizing method of the present invention to carry out the library generation of effective, non-enrichment.
After target sequence in the Single-stranded DNA fragments of the customization oligonucleotide and sex change library with reverse adapter afterbody is annealed, any method as known in the art is used to extend the customization oligonucleotide with reverse adapter afterbody, the method can include but not limited to use the Single-stranded DNA fragments in sex change library as template, utilizes the extension that DNA dependent dna-polymerases carries out.The extension generation with the customization oligonucleotide of reverse adapter afterbody has forward adapter sequence at an end and has the oligonucleotide extension products of reverse adapter sequence at another end.In this embodiment, the customization oligonucleotide with reverse adapter afterbody only can be annealed with the DNA fragmentation in sex change library and extend thereon, this DNA fragmentation comprise the customization oligonucleotide with reverse adapter afterbody for target sequence.As shown in Figure 1, the first primer for forward adapter sequence and the second primer for reverse adapter sequence is used to carry out abatement polymerase chain reaction (PCR) program subsequently, make only to have and at an end, there is forward adapter sequence and the oligonucleotide extension products at another end with reverse adapter sequence can be increased, and therefore enrichment.
Fig. 2 illustrates another embodiment for the abatement PCR enriching method described in Fig. 1, and wherein duplex forward adapter (P1) does not experience breach reparation with the connection of double stranded nucleic acid fragment.Duplex forward adapter (P1) comprises the chain (connection chain be connected with the free 5 ' phosphoric acid on nucleic acid fragment 5 ' end; P1) with chain (the disconnected chain be not connected with nucleic acid fragment 3 ' end; P1rc).In this embodiment, connection chain is connected with 5 ' end of two chains of double stranded nucleic acid fragment, and produces breach or otch between disconnected chain and 3 ' end of double stranded nucleic acid fragment two chains.As shown in Figure 2, after P1 adapter is connected with nucleic acid fragment, carry out sex change when not carrying out breach reparation or filling-in, thus produce the single-chain nucleic acid fragment with frayed end.In this embodiment, the single-chain nucleic acid fragment with frayed end comprises P1 forward adapter sequence at 5 ' end and comprises fragments specific sequence at 3 ' end.In further embodiment, as mentioned above and as shown in Figure 1, can process the single-chain nucleic acid fragment with frayed end further, thus generation has P1 forward adapter sequence at 5 ' end and has the single-chain nucleic acid fragment of different reverse adapter sequences at 3 ' end.
Fig. 4 illustrates another embodiment of the present invention, and it is for efficiently producing the library being included in the nucleic acid fragment each end with different adapter.In this embodiment, method for generation of following sex change library is similar to for the method described in Fig. 1, and this sex change library is included in 5 ' end has single forward adapter sequence and the Single-stranded DNA fragments on 3 ' end with reverse adapter sequence.But, in the diagram, use the oligonucleotide with reverse adapter afterbody, thus this oligonucleotide comprises 3 ' part and the reverse adapter afterbody containing stochastic sequence, wherein this reverse adapter afterbody comprises the reverse adapter sequence different from single forward adapter sequence.As shown in Figure 4, the first primer for single forward adapter sequence and the second primer for reverse adapter sequence can be used to carry out PCR, and wherein the first and second primers comprise flow cell sequence all further.Like this, 5 ' end has single forward adapter sequence and the Single-stranded DNA fragments on 3 ' end with reverse adapter sequence comprises flow cell sequence, this flow cell sequence can be used for the Single-stranded DNA fragments of amplification to be attached to flow cell, so that as United States Patent (USP) 5, and 750,341,6,306,597 and 5,969, described in 119, by carrying out follow-up order-checking by the business-like method of Illumina.
Fig. 3 illustrates the method being included in target nucleic acid sequence in double-strandednucleic acid Insert Fragment or target sequence for enrichment from complex library.In one embodiment, this complex library comprises the Nucleic acid inserts from genome DNA sample.In figure 3, single forward adapter comprises the partial duplex forward adapter containing long-chain A, this long-chain A and short chain B forming section duplex.The chain A of partial duplex adapter comprises restriction enzyme sites further, and chain B is not containing restriction enzyme sites.Chain B comprises blocking groups further, and the 3 ' end of its medium chain B is by modifying with the blocking groups of polymerase extension can be stoped to replace 3 ' OH base.In one embodiment, partial duplex forward adapter is connected with double stranded nucleic acid fragment, thus creates the double-stranded inserts with the partial duplex forward adapter being attached to two ends.In this embodiment, the 5 ' end of the chain B of partial duplex adapter can comprise 5 ' free phosphoric acid, and this 5 ' free phosphoric acid can be connected with 3 ' the free OH be present on of double-stranded inserts or two chains.Sex change subsequently produces and on 5 ' end, comprises sequence A and the strand Insert Fragment comprising chain B on 3 ' end.Primer C for the desired specificities sequence in strand Insert Fragment can anneal with specific sequence, and uses strand Insert Fragment as template, extends with archaeal dna polymerase.In one embodiment, primer C can be sequence specific primers, and for the one or more interested target of method enrichment according to the present invention.In one embodiment, primer C can be random primer.Use archaeal dna polymerase to extend primer C and produce the primer C product extended, this product comprises the sequence with sequence A complementation in its 3 ' end inserting in the double-stranded complex of chain with template, thus creates double-stranded restriction sites between sequence A and its complementary sequence.In one embodiment, this double stranded restriction enzyme recognition site can be cut by the specific restriction enzyme of double-stranded restriction sites, thus produce brachymemma end or the adapter sequence of cutting.Then, use any method of attachment as herein described, the second forward adapter of the duplex adapter D comprising common or routine is connected with cleavage site, thus generation comprises the second forward adapter sequence D an end and in opposite ends, comprises the double-stranded complex of the 3 ' overhang containing chain B.Make comprise the second forward adapter sequence D an end and in opposite ends, comprise the double-stranded complex sex change of the 3 ' overhang containing chain B, and use for first primer of the second forward adapter D and increase for second primer of chain B.By this way, method shown in Fig. 3 can be used for the interested particular sequence of enrichment from complex library, because the method is designed such that the double stranded cleavage site that the second forward primer D can only produce in combination with carrying out restriction enzyme digestion to the double-stranded restriction sites produced between sequence A and its complementary sequence after primer C extends.As described in, primer C can for the target sequence in the single or multiple one or more Insert Fragments be present among multiple Insert Fragment.In addition, be combined by the target sequence be designed to by primer C on the chain being present in the Insert Fragment among multiple Insert Fragment or another chain, the method can make chain have specificity.
Except as otherwise noted, otherwise genetics used herein, molecular biology, biological chemistry and nucleic acid term and symbol adopt term and the symbol of standard treatise in this field and textbook, such as Kornberg and Baker, DNAReplication, the second edition (W.H.Freeman, NewYork, 1992); Lehninger, Biochemistry, the second edition (WorthPublishers, NewYork, 1975); Strachan and Read, HumanMolecularGenetics, the second edition (Wiley-Liss, NewYork, 1999); Eckstein, editor, OligonucleotidesandAnalogs:APracticalApproach (OxfordUniversityPress, NewYork, 1991); Gait, editor, OligonucleotideSynthesis:APracticalApproach (IRLPress, Oxford, 1984); Etc..
input nucleic acid
Input can be nucleic acid.In one embodiment, input can be DNA.In one embodiment, input nucleic acid can be compound DNA, such as double-stranded DNA, genomic dna or from more than an organic hybrid dna.In one embodiment, input can be RNA.In one embodiment, RNA can use the standard technique in this area to obtain and purifying, and comprise the RNA of purifying or non-purified form, it includes but not limited to mRNA, tRNA, snRNA, rRNA, retrovirus, little non-coding RNA, Microrna, polysome RNA, premessenger RNA, introne RNA, viral RNA, acellular RNA and fragment thereof.Non-coding RNA or ncRNA can comprise snoRNA, Microrna, siRNA, piRNA and long ncRNA.In one embodiment, inputting nucleic acid can be cDNA.CDNA can be produced by RNA such as mRNA.CDNA can be strand or double-strand.Input DNA can be the DNA of the specific species such as such as people, rat, mouse, other animals, specified plant, bacterium, algae, virus.Input mixture also can from the genomic mixture of the such as different plant species such as host-pathogen, bacterial population.Input DNA can be the cDNA prepared by the genome mixture of different plant species.Or input nucleic acid can from synthesis source.Input DNA can be Mitochondrial DNA.Input DNA can be Cell-free DNA.Cell-free DNA can obtain from such as serum or plasma sample.Input DNA can comprise one or more karyomit(e).Such as, if input DNA is from the mankind, then DNA can comprise karyomit(e) 1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22, one or more in X or Y.DNA can from linear or ring-type genome.DNA can be plasmid DNA, cosmid DNA, bacterial artificial chromosome (BAC) or yeast artificial chromosome (YAC).Input DNA can from more than body or organism one by one.Input DNA can be double-strand or strand.Input DNA can be a chromatinic part.Input DNA can be relevant to histone.
In some embodiments, the oligonucleotide in target sequence region that target is selected is designed to and single-chain nucleic acid target hybridization.In one embodiment, the oligonucleotide in target sequence region that target is selected is designed to hybridize with Single stranded DNA targets.When inputting nucleic acid samples and comprising genomic dna or other double-stranded DNAs, first can make input nucleic acid denaturing samples, to make target become strand, and allow the target sequence area hybridization of oligonucleotide and expectation.In these embodiments, method and composition described herein can allow regiospecificity enrichment and amplification target sequence region.In some embodiments, other double-stranded DNAs can be the double-strand cDNA produced by the first and second chain synthesis of one or more target RNA.
In other embodiments, the oligonucleotide in target sequence region that target is selected is designed to hybridize with this double-strand nucleic acid target when double-strandednucleic acid unchangeability.In other embodiments, the oligonucleotide in target sequence region that target is selected be designed to when dsDNA unchangeability with this double-stranded DNA target hybridization.In these embodiments, the oligonucleotide in target sequence region that target is selected is designed to form triple helix (triplex) in selected target sequence region.When sample double-stranded nucleic acid does not have sex change in advance, the hybridization in oligonucleotide and target double chain DNA sequence region can be carried out.In this type of embodiment, method and composition described herein can allow the regiospecificity enrichment in target sequence region and chain specific enrichment and amplification.This method can be used for the copy producing object chain specific sequence region from composite nucleic acid, and without the need to making the dsDNA sex change of input DNA, therefore, it is possible to the multiple target sequence regions of concentration and analysis in original composite nucleic acid sample.The method can be used for the research and analysis that original position is carried out, can research and analysis unicellular or minimum, complex gene group DNA in clearly defined cell colony set, and allow to analyze complex gene group DNA when not destroying chromatin Structure.
" target nucleic acid sequence " or " target sequence " is the subject polynucleotide sequence of expecting to carry out it enrichment as used herein.In its actual sequence, target sequence can be known or the unknown.Usually, " template " is the polynucleotide comprising target nucleic acid sequence as used herein.Term " target sequence ", " target nucleic acid sequence ", " target nucleotide sequences ", " target area " or " target sequence " and the commutative use of variant thereof.
oligonucleotide of the present invention
As used in the present invention, term " oligonucleotide " refers to polynucleotide chain, is normally less than the length of 200 residues, and modal is the length of 15-100 Nucleotide, and is intended to comprise longer polynucleotide chain.Oligonucleotide can be strand or double-strand.As used in the present invention, term " oligonucleotide " can exchange with term " primer " and " adapter " and use.
As used herein, term " hybridization (hybridization) "/" hybridization (hybridizing) " and " annealing " commutative use and refer to the pairing of complementary nucleic acid.
" primer " can refer to the nucleotide sequence usually with 3 ' free hydroxyl as the term is employed herein, it can be hybridized with template (as one or more target polynucleotides, one or more target DNAs, one or more targets RNA or primer extension product), and also can promote and being polymerized of the polynucleotide of template complementation.Primer can be such as oligonucleotide.Can also be, such as, the sequence of template is (as primer extension product, or the template segments produced after RNase [i.e. RNaseH] cutting board-DNA mixture), this sequence can with the sequence hybridization in template itself (such as, as hairpin loop), and the polymerization of Nucleotide can be promoted.Therefore, primer can be exogenous (such as, adding) primer or endogenous (such as, template segments) primer.Primer can containing the non-hybridization sequences forming primer tail.Even if the sequence of primer not with target complete complementary, its still can with target hybridization.
Primer of the present invention normally in the extension undertaken by polysaccharase along polynucleotide template, such as PCR, SPIA or cDNA synthesis in adopt oligonucleotide.Oligonucleolide primers can be the synthetic polyribonucleotides of strand, contains the sequence can hybridized with target polynucleotide sequence at its 3 ' end.Usually, 3 ' region of the primer can hybridized with target nucleic acid and sequence or primer binding site have at least 80%, the complementarity of preferably 90%, more preferably 95%, most preferably 100%.
As used herein " complementary " can refer to the whole of sequence or only with the complementarity of a part for sequence.Specific oligonucleotide primers can the stringency for hybridizing this Oligonucleolide primers should be made will to stop too much random nonspecific hybridization by the few nucleotide in hybridization sequences.Usually, few nucleotide in the hybridization portion of Oligonucleolide primers is by equally large for the restriction sequence on the target polynucleotide of at least hybridizing with this Oligonucleolide primers, namely, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least about 20, and about 6 to about 10 or 6 to about 12 or 12 to about 200 Nucleotide, usually about 20 to about 50 Nucleotide usually.In general, target polynucleotide is greater than Oligonucleolide primers or previously described primer.
In some cases, the identity of the target polynucleotide sequence studied is known, and accurately can synthesize according to the antisense sequences of aforementioned target polynucleotide sequence can the specific oligonucleotide of hybridization sequences or primer.In some embodiments, use multiple sequence specific oligonucleotide or primer to come to hybridize with a large amount of target genome areas, thus allow the selective enrichment of target area.When genome area may be grown very much, multiple oligonucleotide design can be become hybridize from the different sequence areas in target genome area.In other embodiments, when target polynucleotide sequence is unknown, oligonucleotide or primer can hybridization sequences be stochastic sequence.As described herein, the oligonucleotide or the primer that comprise stochastic sequence can be called as " random primer " or " random oligonucleotide ".In one embodiment, the oligonucleotide of the present invention can hybridized with target sequence or primer can comprise be designed to multiple (such as 2,3,4, about 6,8,10,20,40,80,100,125,150,200,250,300,400,500,600,800,1000,1500,2000,2500,3000,4000,5000,6000,7000,8000,10,000,20,000,25,000 kind or more to plant) primer of target sequence hybridization or the mixture of oligonucleotide.In some cases, described multiple target sequence can comprise one group of correlated series, stochastic sequence, whole transcript profile or its part (such as, major part), or any sequence is in groups as mRNA.In some embodiments, primer can be directed to the known array existed in the adapter used in the present invention as herein described.In this embodiment, primer can comprise the primer in groups in each group containing one or more primer, and wherein often organizing primer can for different adapter.
Tail primer or oligonucleotide can be adopted in certain embodiments of the invention.Usually, tail primer is had to comprise the 3 ' part can hybridized with one or more target polynucleotides and the 5 ' part can not hybridized with one or more target polynucleotides.Usually, under the condition having the interfertile 3 ' part of tail primer and one or more target polynucleotides to hybridize, not interfertile 5 ' part is not hybridized with these one or more target polynucleotides.In some embodiments, not interfertile 5 ' part comprises adapter sequence.In some embodiments, not interfertile 5 ' part comprises adapter sequence that is common or routine.In some embodiments, not interfertile 5 ' part comprises adapter sequence that is common or routine, and this adapter sequence is different from or is different from the sequence of other adapters used in the present invention.In some embodiments, not interfertile 5 ' part comprises promotor specific sequence.Usually, promotor specific sequence comprises single-stranded DNA sequence region, and this region can be transcribed by mediate rna under double chain form.The example of promotor specific sequence is known in the art, and includes but not limited to T7, T3 or SP6RNA polymerase promoter sequence.When utilizing archaeal dna polymerase to be extended with tail primer, the primer extension product having and comprise the 5' part limiting sequence can be produced.Then this primer extension product can have the second primer to anneal with it, and this second primer can utilize archaeal dna polymerase to extend to produce the double-stranded products at one end comprising and limit sequence.One or more have the not interfertile 5 ' part of tail primer to comprise in some embodiments of promotor specific sequence wherein, and the structure at one end comprising the double-stranded products limiting sequence produces can the mediate rna double-stranded promoter sequence of transcribing.In some embodiments, hybridize to produce double-stranded promoter sequence with the oligonucleotide of the sequence of promotor specific sequence complementation and promotor specific sequence by making to comprise.In some embodiments, then can produce single stranded RNA by carrying out rna transcription to the downstream sequence of double-stranded promoter after the formation of double-stranded promoter, usually including but not limited to that ribonucleotide triphosphate (rNTP) and all of DNA dependent rna polysaccharase must be carried out in the reaction mixture of component comprising.There is tail primer can comprise DNA, RNA or DNA and RNA.In some embodiments, be made up of DNA tail primer.
Composite primer can be adopted in certain embodiments of the invention.The primer that composite primer is made up of RNA and DNA part.In some respects, composite primer can be comprise such as 3'-DNA part and 5'-RNA partly have tail composite primer.Have in tail composite primer at this, its all or part of comprise the 3' part of DNA and polynucleotide complementary; And its all or part of to comprise the 5' part of RNA complementary with this polynucleotide, and under this has the 3' part of tail composite primer and the condition of polynucleotide target hybridization not with this multi-nucleotide hybrid.When being extended with tail composite primer with archaeal dna polymerase, the primer extension product having and comprise the 5'-RNA part limiting sequence can be produced.Then this primer extension product can have the second primer to anneal with it, and this second primer can utilize archaeal dna polymerase to extend to produce the double-stranded products at one end having and comprise the RNA/DNA heteroduplex limiting sequence.To produce the double-stranded DNA with 3' single-stranded overhang, this double-stranded DNA all respects used in the present invention, can comprise and allow to use composite amplification primer to carry out isothermal duplication from selectivity cutting RNA part part heteroduplex.
" random primer " can be the primer usually comprising a kind of like this sequence as used herein: this sequence not necessarily designs based on the specific or specific sequence in sample, but designs based on the statistical expection (or empirical observation) that the sequence of random primer can be hybridized with one or more sequences (under a given set condition) in sample.Random primer normally comprises oligonucleotide or the oligonucleotide colony of stochastic sequence, the Nucleotide of the given position wherein on oligonucleotide can be the selected arbitrarily group (only three kinds in such as four kinds of Nucleotide, or in four kinds of Nucleotide only two kinds) of any Nucleotide in four kinds of Nucleotide or four kinds of Nucleotide.In some cases, all positions of oligonucleotide or oligonucleotide colony can be two or more Nucleotide any.In other cases, an only part for oligonucleotide, such as specific region, comprising can be the position of arbitrarily two or more bases.In some cases, the oligonucleotide part comprising the position that can be two or more bases arbitrarily has the length of about 5,6,7,8,9,10,11,12,13,14 or an about 15-20 Nucleotide.In some cases, random primer can include tail primer, and this has tail primer to have to comprise the 3' district of stochastic sequence and as the 5' district of non-hybridization sequences comprising specificity nonrandom sequences.3' district also can comprise the stochastic sequence with the areas combine comprising poly-T sequence.The sequence of random primer (or its complement) can be or can not be naturally occurring, or may reside in or can not be present in the sequence pond in interested sample.As known in the art, " random primer " also can refer to such primer: its for Joint Designing be with expect and/or the member of primer colony (multiple random primer) that a large amount of target sequence is hybridized.Random primer can the multiple site on nucleotide sequence be hybridized.The use of random primer provides a kind of method for generation of the primer extension product with target polynucleotide or target nucleic acid sequence complementation, and the method does not need the exact nucleotide sequence understanding target in advance.In some embodiments, a part for primer is random, and another part of primer comprises the sequence of restriction.Such as, in some embodiments, the 3' part of primer will comprise stochastic sequence, and the 5' of primer part comprises the sequence of restriction.In some embodiments, the 3' random partial of primer will comprise DNA, and the 5' qualifying part of primer will comprise RNA, and in other embodiments, 3' and 5' part all will comprise DNA.In some embodiments, 5' part will comprise the sequence of restriction, and 3' part will comprise the poly-dT sequence can hybridized with a large amount of RNA (as all mRNA) in sample.In some embodiments, " random primer " or the primer comprising the random sequence produced comprise the set of primer, this primer set comprises one or more Nucleotide being selected from two or more different IPs thuja acids at random, and all possible combined sequence of the Nucleotide of Stochastic choice can be presented in this set.In some embodiments, the generation of one or more random primers does not comprise and to get rid of from the possible combined sequence of the random partial of one or more random primers or to select the step of some sequence or nucleotide combination.
In one embodiment, oligonucleotide of the present invention can be have tail oligonucleotide.In one embodiment, 5 '-tail can comprise RNA, and can not hybridize with the RNA in sample.In one embodiment, 5 '-tail can comprise DNA, and can not with the DNA hybridization in sample.In one embodiment, 5 '-tail can comprise adapter, and this adapter can not be hybridized with from containing the DNA in the sample of nucleic acid and/or nucleic acid fragment.In one embodiment, 5 '-tail can comprise adapter sequence, and this adapter sequence can not be hybridized with from containing the DNA in the sample of nucleic acid and/or nucleic acid fragment.In some embodiments, 5 '-tail can comprise common adapter sequence, and this common adapter sequence can not be different from DNA hybridization any other adapter or adapter sequence of using in the inventive method as herein described.In some embodiments, 5 '-tail can comprise identifier.In some embodiments, this identifier can comprise bar code sequence.In some embodiments, 5 '-tail can comprise common adapter sequence, and this common adapter sequence can not be hybridized with DNA and bar code sequence.
" adapter " refers to the oligonucleotide of known array as the term is employed herein, and the connection of itself and target polynucleotide or target polynucleotide chain makes it possible to produce ready (amplification-ready) product of the amplification of this target polynucleotide or target polynucleotide chain.Before adapter adds, target polynucleic acid molecules can fragmentation or do not carry out fragmentation.
It is contemplated that the various adapter designs of the ready product of amplification being suitable for producing target sequence area/chain.Such as, two chains of adapter can be self-complementary, non-complementary or partial complementarity.The common trait of the adapter described in Fig. 3 of the present invention is partial duplex design, and wherein two chains of adapter have different length, have complementary region and outburst area at 5 ' end.5 ' end of the long-chain of partial duplex adapter comprises for the unique site of nucleic acid modifying enzyme as restriction enzyme, and this site is not present in the short chain of this duplex adapter.3 ' end of short chain adapter is replaced 3 ' OH group by blocking groups such as dideoxy nucleotide (ddCMP, ddAMP, ddTMP or ddGMP) and is modified, to stop polymerase extension.
In some embodiments of the present invention, adapter comprises extra identifier, i.e. bar code sequence.As used herein, term " barcode " refers to the known nucleic acid sequence allowing some features identifying the polynucleotide be associated with barcode.In some embodiments, the feature of the polynucleotide that will identify is the sample deriving these polynucleotide.In some embodiments, barcode is at least the length of 3,4,5,6,7,8,9,10,11,12,13,14,15 or more Nucleotide.In some embodiments, barcode be shorter in length than 10,9,8,7,6,5 or 4 Nucleotide.In some embodiments, each barcode in multiple barcode at least on 3 nucleotide positions, such as, is at least different from other barcodes each in described multiple barcode on 3,4,5,6,7,8,9,10 or more positions.In some embodiments, compared with the barcode be associated with some polynucleotide and the barcode that is associated with other polynucleotide, there is different length.Usually, barcode has enough length and comprises enough different sequences to allow to differentiate sample based on the barcode be associated with sample.In some embodiments, forward and reverse adapter all comprise at least one in multiple bar code sequence.In some embodiments, first, second and/or the 3rd adapter comprise at least one in multiple bar code sequence.In some embodiments, each reverse adapter comprises at least one in multiple bar code sequence, and each bar code sequence in wherein said multiple bar code sequence is different from other bar code sequences each in described multiple bar code sequence.In some embodiments, the first adapter and the second adapter all comprise at least one in multiple bar code sequence.In some embodiments, for the barcode of the second adapter oligonucleotide independently selected from the barcode for the first adapter oligonucleotide.In some embodiments, the first adapter oligonucleotide and the second adapter oligonucleotide with barcode match, thus make this comprise identical or different one or more barcodes to adapter.In some embodiments, method of the present invention comprises the sample that the bar code sequence connected based on target polynucleotide differentiates to derive this target polynucleotide further.Usually, barcode comprises such nucleotide sequence, and when being connected to target number Nucleotide, it can be used as the marker of the sample deriving target polynucleotide.
Recently, made at adapter design aspect the improvement that many minimizing adapter dimers occur.These improve to comprise and use nucleotide analog and structurized oligonucleotide, and allow the oligonucleotide using higher concentration in ligation.The adapter of the higher concentration in ligation enables researchist produce high-quality library from few to 150 genome copies.The connection of the end of adapter and DNA fragmentation, is particularly applicable to implementing method of the present invention with the connection of the end of those fragments containing target area.Multiple mode of connection is contemplated based on the selection of nucleic acid modifying enzyme and the double-stranded DNA cutting of generation.Such as, when producing the blunt-end product containing target region/sequence, it may be suitable that flush end connects.Or, cut the specific restriction enzyme of use known array, thus when causing producing the cleavage site with known array overhang, the suitable end of adapter can be designed to be able to make the cleavage site in adapter and target sequence region to hybridize, and connects subsequently.Effectively and the rapid reagent that is connected and method for adapter is commercially available acquisition and is as known in the art.
nucleic acid modifying enzyme
Nucleic acid (NA) modifying enzyme can be DNA specificity modifying enzyme.NA modifying enzyme can be selected according to the specificity for double-stranded DNA.This enzyme can be duplex specific endonuclease, flush end cuts restriction enzyme (blunt-endfrequentcutterrestrictionenzyme) or other restriction enzymes frequently.The example of flush end nickase comprises DraI or SmaI.NA modifying enzyme can be the enzyme provided by NewEnglandBiolabs.NA modifying enzyme can be target-seeking endonuclease (target-seeking endonuclease can be the endonuclease of the recognition sequence without strict difinition).NA modifying enzyme can be otch endonuclease (otch endonuclease can be can only the endonuclease of a DNA chain in cutting double-stranded DNA substrate).NA modifying enzyme can be high-fidelity endonuclease (high-fidelity endonuclease can be the endonuclease of through engineering approaches, and it has " star activity " lower than the wild-type form of this endonuclease).
In a preferred embodiment, NA modifying enzyme is the specific DNA modification enzyme of sequence and duplex.
dNA dependent dna-polymerases
Method according to the present invention the extension of primer or oligonucleotide can be realized for the DNA dependent dna-polymerases in method and composition of the present invention.In one embodiment, preferred DNA dependent dna-polymerases can be the polysaccharase that can extend nucleic acid primer under the existence of RNA or cDNA template.The exemplary DNA dependent dna-polymerases being applicable to method of the present invention includes but not limited to that tool is with or without the Klenow polysaccharase of 3'-exonuclease activity, BstDNA polysaccharase, Bca polysaccharase, φ 29DNA polysaccharase, Vent polysaccharase, DeepVent polysaccharase, Taq polysaccharase, T4 polysaccharase and e. coli dna polymerase 1, its derivative, or the mixture of polysaccharase.In some cases, polysaccharase does not comprise 5'-exonuclease activity.In other cases, polysaccharase comprises 5' exonuclease activity.In some cases, primer of the present invention or oligonucleotide extension can use the polysaccharase such as Bst polysaccharase comprising strong strand-displacement activity to carry out.In other cases, primer extension of the present invention can use and comprise weak or without strand-displacement activity polysaccharase and carry out.Those skilled in the art will recognize that the merits and demerits of usage chain substitute activity in primer extension procedures, and estimate which polysaccharase can provide strand-displacement activity (such as, see, NewEnglandBiolabs polysaccharase).
amplification method
Method as herein described, composition and test kit can be used for producing the ready product of amplification, for downstream application, as extensive parallel order-checking (i.e. new-generation sequencing method), the generation with the library of the target sequence region colony of enrichment or hybridization platform.Amplification method is well known in the art.Suitable amplified reaction can be exponential or isothermal and can comprise any DNA amplification reaction, include but not limited to polymerase chain reaction (PCR), strand displacement amplification (SDA), linear amplification, multiple displacement amplification (MDA), rolling circle amplification (RCA), single primer isothermal duplication (SPIA, see, such as U.S. Patent number 6,251,639), Ribo-SPIA, or its combination.In some cases, can carry out under restricted condition for providing the amplification method of template nucleic acid, severally amplification is taken turns (such as to make only to carry out, 1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29,30 to take turns), such as usual generation for cDNA is carried out.The wheel number of amplification can be that about 1-30,1-20,1-15,1-10,5-30,10-30,15-30,20-30,10-30,15-30,20-30 or 25-30 take turns.
PCR is a kind of amplification in vitro program, it is based on sex change, Oligonucleolide primers annealing and the recirculation of primer extension of being undertaken by thermophilic Template Dependent polynucleotide polysaccharase, thus causes flank to be the exponential growth of the expectation sequence copy of the polynucleotide analyte of primer.Location can be different from two that the opposite strand of DNA is annealed PCR primer, with the template strand making the polymerase catalysed extension products of a primer can be used as another primer, thus cause the accumulation of discrete double-stranded segment, the length of this fragment by Oligonucleolide primers 5' end between distance limited.
The preformed nucleic acid probe that LCR uses ligase enzyme to be connected into pairs.Every bar complementary strand (if present) of these probes and nucleic acid analyte is hybridized, and adopt ligase enzyme to be combined by often pair of probe, thus produce two templates that can be used to repetition (reiterate) specific nucleic acid sequence in next one circulation.
SDA (people such as Westin, 2000, NatureBiotechnology, 18,199-202; The people such as Walker, 1992, NucleicAcidsResearch, 20,7,1691-1696) be a kind of isothermal amplification technique, it is based on restriction endonuclease as HincII or BsoBI makes the unmodified chain of half thiophosphoric acid form of its recognition site produce the ability of otch, and exonuclease-deficient archaeal dna polymerase extends 3' end as Klenowexominus polysaccharase or Bst polysaccharase in incision and makes the ability of downstream DNA chain displacement.Exponential amplification causes by reacting in conjunction with sense and antisense, and wherein from the target having the chain of adopted action displacement to react as antisense, vice versa.
Aspects more of the present invention utilize the linear amplification of nucleic acid or polynucleotide.Linear amplification typically refers to a kind of so method, and it comprises one or more copies of the complement of the only chain forming nucleic acid or polynucleotide molecule (being generally nucleic acid or polynucleotide analyte).Therefore, essential difference between linear amplification and exponential amplification is: in a rear method, product is as the substrate for the formation of more voluminous thing, and in last method, homing sequence is the substrate for the formation of product, but copying of reaction product and starting template is not substrate for generation of product.In linear amplification, the product amount of formation as the time linear function and increase, be different from the exponential amplification that the product amount wherein formed is the exponential function of time.
In some embodiments, amplification is exponential, such as, in the enzymatic amplification undertaken by the specific double-strand sequence of polymerase chain reaction (PCR) to DNA.In other embodiments, amplification method is linear.In other embodiments, amplification method is isothermal.
downstream application
An importance of the present invention is, method and composition disclosed herein can efficiently and cost effectively for downstream analysis, as new-generation sequencing or hybridization platform, there is the loss of minimum target biomaterial.Method of the present invention also can be used for target selectivity genome area (such as, the analysis of SNP or other diseases mark) and can with the analysis of the genetic information of the genome area of target selectivity regional interaction.
order-checking
Such as, method of the present invention for by as U.S. Patent number 5,750,341,6,306,597 and 5,969, described in 119, to be undertaken checking order by the business-like method of Illumina be useful.Usually, double-stranded segment polynucleotide by method of the present invention preparation to produce the nucleotide sequence of the amplification marked at (such as (A)/(A ')) or two end (such as (A)/(A ') and (C)/(C ')) places.In some cases, the single-chain nucleic acid marked in one or two end is increased by method of the present invention (such as, by SPIA or linear PCR).Then the nucleic acid denaturation will obtained, and the internal surface polynucleotide of single stranded amplification being connected to randomly flow cell passage.Add unlabelled Nucleotide to start solid phase bridge amplification to produce the dense cluster of double-stranded DNA.In order to start the first base order-checking circulation, add the reversible terminator of four kinds of marks, primer and archaeal dna polymerase.After laser excitation, imaging is carried out to the fluorescence from each bunch on flow cell.Then the identity of first base of each bunch is recorded.Carry out order-checking circulation often to determine this fragment sequence to next base.
In some embodiments, method of the present invention can be used for preparing target polynucleotide, is checked order by the business-like connection sequence measurement of AppliedBiosystems (such as, SOLiD checks order) for passing through.In other embodiments, these methods can be used for preparing target polynucleotide, carry out synthesis order-checking to use by the business-like method of 454/RocheLifeSciences, these methods include but not limited to people such as Margulies, Nature (2005) 437:376-380 (2005) and U.S. Patent number 7,244,559,7,335,762,7,211,390,7,244,567,7,264,929 and 7,323, the method and apparatus described in 305.In other embodiments, these methods can be used for preparing target polynucleotide, so that as Application U.S. Serial No 11/167,046 and U.S. Patent number 7,501,245,7,491,498,7,276,720 and U.S. Patent Application Publication No. US20090061439, described in US20080087826, US20060286566, US20060024711, US20060024678, US20080213770 and US20080103058, by being checked order by the business-like method of HelicosBioSciencesCorporation (Cambridge, Mass.).In other embodiments, these methods can be used for preparing target polynucleotide, so that as U.S. Patent number 7, and 462,452,7,476,504,7,405,281,7,170,050,7,462,468,7,476,503,7,315,019,7,302,146,7,313,308 and U.S. Application Publication No US20090029385, US20090068655, US20090024331 and US20080206764 described in, by being checked order by the business-like method of PacificBiosciences.
Another example of the sequencing technologies that can use in the method for the present invention provided is nanoporous order-checking (such as, see, SoniGV and MellerA. (2007) ClinChem53:1996-2001).The aperture of nanoporous can be diameter be about 1 nanometer.The applying of the electromotive force of the submergence of nanoporous in transfer fluid and transadmittance metre hole can cause slight electric current by the conduction of nanoporous due to ion.The size of the magnitude of current to nanoporous of flowing is responsive.Along with DNA molecular is by nanoporous, each Nucleotide on DNA molecular is with different throttle nanoporouss.Therefore, when DNA molecular is by representing the reading to DNA sequence dna by the change of the electric current of nanoporous during nanoporous.
Another example of the sequencing technologies that can use in the method for the present invention provided is the semi-conductor order-checking (such as, using IonPersonalGenomeMachine (PGM)) provided by IonTorrent.The technology of IonTorrent can use the semi-conductor chip with multiple layer (such as, having the layer in the hole of micromachined, ion-sensitive layer and ionization sensor layer).Can nucleic acid be added in hand-hole, such as, the clonal population of single nucleic acid can be attached on single pearl, and can by this pearl introduction hole.In order to be enabled in the order-checking of the nucleic acid on pearl, can by the deoxyribonucleotide of a type (such as, dATP, dCTP, dGTP or dTTP) introduction hole.When being mixed one or more Nucleotide by archaeal dna polymerase, in hole, discharge proton (hydrogen ion), this can be detected by ionization sensor.Then can washing semi-conductor chip, and different deoxyribonucleotides can be utilized to repeat this process.Can check order to multiple nucleic acids in the hole of semi-conductor chip.Semi-conductor chip can comprise chemosensitivity field-effect transistor (chemFET) array with check order to DNA (such as, as described in U.S. Patent Application Publication No. 20090026082).The change of mixing electric current by with chemFET measure of one or more triphosphoric acids in 3 ' end of sequencing primer to new nucleic acid chain detects.Array can have multiple chemFET sensor.
genetic analysis
Method of the present invention can be used for target selectivity genome area and can with the analysis of the genetic information of the genome area of target selectivity regional interaction.Amplification method disclosed herein can be used for the device for genetic analysis known in the art, test kit and method, such as but not limited at U.S. Patent number 6, and 449,562,6,287,766,7,361,468,7,414,117,6,225,109 and 6,110, in 709 find those.In some cases, amplification method of the present invention can be used for the target nucleic acid increased for DNA hybridization research, to determine the presence or absence of polymorphism.Polymorphism or allelotrope can to disease or situation as relevant in inherited disease.In other cases, polymorphism can be relevant to the susceptibility of disease or situation, such as, to habituation, polymorphism that degeneration is relevant with age related situation, cancer etc.In other cases, polymorphism can be relevant to beneficial characteristics, this beneficial characteristics is such as that coronary artery health strengthens, or the resistibility of disease to such as HIV or malaria and so on, or the resistibility to the such as degenerative disease of osteoporosis, Alzheimer's disease or dementia and so on.
test kit
Any composition as herein described all can be included in test kit.In limiting examples, the test kit be in suitable container comprises: one or several adapters, for connecting, one or more Oligonucleolide primers of primer extension and amplification and reagent.Test kit also can comprise the device for purifying, such as pearl suspension.
Component maybe can be placed on comprising at least one bottle, test tube, flask, bottle, syringe and preferably suitable decile other containers in the inner by the container of test kit usually.When existing in test kit more than a kind of component, this test kit usually also by containing second, third container maybe can by extra component separated other extra containers wherein.But, the various combinations of component can be comprised in a reservoir.
When providing the component of test kit in one or more liquors, this liquor can be the aqueous solution.But the component of test kit can be used as dried powder to be provided.When reagent and/or component provide as dry powder, rebuild this powder by adding suitable solvent.
Test kit will preferably include about using reagent constituents and using the explanation of any other reagent be not included in this test kit.Explanation can comprise the version that can implement.
In one aspect, the invention provides the test kit being included in any one or more elements disclosed in aforesaid method and composition.In some embodiments, test kit comprises composition of the present invention in one or more container.In some embodiments, the invention provides the test kit comprising adapter as herein described, primer and/or other oligonucleotide.In some embodiments, test kit comprise further following in one or more: (a) DNA ligase; (b) DNA dependent dna-polymerases; (c) RNA dependent dna-polymerases; (d) forward adapter; (e) one or more contain the oligonucleotide of reverse adapter sequence; And (f) is applicable to one or more buffered soln of the one or more elements be included in described test kit.Adapter, primer, other oligonucleotide and reagent may be, but not limited to, any adapter mentioned above, primer, other oligonucleotide and reagent.The element of test kit can provide with (but being not limited to) any amount mentioned above and/or combination (such as in identical test kit or identical container) further.Test kit can comprise the additional agents used according to the inventive method further, all reagent as described above.Such as, test kit can comprise the first forward adapter (it is partial duplex adapter as described herein), the second forward adapter and have specific nucleic acid modifying enzyme for the restriction existed in the first forward adapter and/or cleavage site.Test kit element can provide in any suitable container including but not limited to test tube, bottle, flask, bottle, ampoule, syringe etc.Reagent can provide with the form that can directly use in the methods of the invention, or provides to need to prepare the form that such as freeze-dried preparation is rebuild before use.Reagent can provide for disposable equal portions form, or repeatedly uses the form of the stock solution of (such as in some reactions) to provide obtaining.
In one embodiment, the explanation that test kit comprises multiple forward adapter oligonucleotide and uses about it, wherein each described forward adapter oligonucleotide comprises at least one in multiple bar code sequence, and each bar code sequence in wherein said multiple bar code sequence is different from other bar code sequences each in described multiple bar code sequence at least three nucleotide positions.The forward adapter comprising different bar code sequence can provide separately or combine from the one or more extra forward adapter with different bar code sequence and provide.In some embodiments, test kit can comprise multiple first and second forward adapter oligonucleotide.Second forward adapter oligonucleotide can separate from one or more first forward adapter and/or one or more the second different adapter or combine to be provided.The combination of the first and second forward adapters can be provided according to aforesaid combination.In some embodiments, test kit can comprise multiple oligonucleotide containing reverse adapter sequence.In one embodiment, test kit can comprise multiple oligonucleotide containing reverse adapter sequence, wherein multiplely comprises further and the sequence of desired specificities target complement sequence that exists in nucleic acid containing each in the oligonucleotide of reverse adapter sequence.In one embodiment, test kit can comprise multiple oligonucleotide containing reverse adapter sequence, and wherein each containing in the oligonucleotide of reverse adapter sequence multiple comprises stochastic sequence further.In one embodiment, test kit comprises multiple oligonucleotide with reverse adapter sequence and the explanation used about it, each having in the oligonucleotide of reverse adapter sequence wherein said comprises at least one in multiple bar code sequence, and each bar code sequence in wherein said multiple bar code sequence is different from other bar code sequences each in described multiple bar code sequence at least three nucleotide position places.The oligonucleotide with reverse adapter sequence comprising different bar code sequence can provide separately or combine from one or more extra oligonucleotide with reverse adapter sequence with different bar code sequence and provide.
based on the product of the inventive method
Product based on the inventive method can by the applicant with trade(brand)name carry out commercialization. it is the trade mark of NuGENTechnologies, Inc..
Embodiment
Embodiment 1-passes through the selective enrichment of bacterial 16 S ribosomal DNA sequence dna to the sign of Human Oral Cavity microorganism group.
sample nucleic
According to the specification sheets of manufacturers, use OMNIgene-DISCOVER sample collecting test kit (DNAGenotek) separate microorganism genomic dna from people's saliva.Then by supersound process, the DNA fragmentation of extraction is changed into the mean length of 400bp, and use AgencourtAMPureXP pearl (BeckmanCoulterGenomics) to carry out purifying.
contrast and be connected with the generation in the test library of forward adapter
NuGENOvationUltralow library system (NuGENTechnologies) is utilized to produce two new-generation sequencing libraries by the sample of 100ng purifying.The first library is prepared, i.e. the contrast of non-enrichment according to the recommendation of manufacturers.The following identical library construction Kit improved is used to produce second " test " library, namely for the input of downstream enriching step.In brief, make DNA flat end, and prepare to connect under the Typical end described in test kit repairs reaction conditions.Fragment is only connected to forward adapter subsequently.As shown in Figure 2, connect and each end of forward adapter and each DNA fragmentation is linked together, thus produce single-stranded nick on opposite strand.Carry out adapter to fill, produce when therefore there is spacer end on each Insert Fragment and connect product.
At least the connection product of 100bp length is by carrying out purifying with the selective binding of AgencourtAMPureXP pearl, and carries out enrichment process subsequently.
amplification
The rDNA fragment coming self-test library two different steps carry out selective amplification: 1) gene-specific primer extends; And 2) use the PCR of general adapter sequence.Primer extension procedures implemented by the oligonucleotide that use comprises 3 ' gene specific region and 5 ' common region, and wherein this 5 ' common region contains a part for the reverse adapter sequence of Illumina.Compare the rrna operon from 40 kinds of different bacterium kinds by use ClustalW Multiple sequence alignments program (European Bioinformatics institute (EuropeanBioinformaticsInstitute)), select the total 16S sequence forming gene specific section.Synthesize following oligonucleotide and mix with equimolar ratio example, this oligonucleotide represents each in the block of 18 high conservatives identified in 16S genomic gene seat.
Primer extension probes pond and the test dna library (as above) containing forward adapter and the main mixture (QIAGEN, USA) of HotStarTaqPCR containing damping fluid, dNTP and heat activated Taq DNA polymerase are mixed.This solution is placed in thermal cycler, be heated to 95 DEG C keep 15 minutes to activate polysaccharase, and be cooled to 70 DEG C maintenances 5 minutes to make 16S primer and DNA Insert Fragment anneal and to extend to forward adapter site.Add the amplimer that can be combined with forward and reverse adapter site.By using 3 step temperature course, (94 DEG C keep 30 seconds, 60 DEG C keep 30 seconds, and 72 DEG C keep 1 minute) PCR that carries out 25 circulations carried out to the fragment containing forward (test library) and reverse (5 ' common region on 16S primer) adapter and respective general priming site selection.Use AMPureXP pearl purified pcr product and analyze with 2100 biological analysers (AgilentTechnologies).
order-checking and data analysis
Use MiSeq system (Illumina), the test library for contrast and enrichment obtains single end sequencing reading (reads) that length is 100nt.Software processes raw sequencing data and is mapped to (mappedto) ribosomal RNA database to use Illumina base to judge.By the sequence mapping that do not line up with bacterium rRNA to the mankind and bacterium full-length genome reference sequences.By being that the per-cent of total mapping reading in contrast and test sample determines enrichment times by the digital computation of rRNA reading.
Embodiment 2-passes through the selective enrichment of bacterial 16 S ribosomal DNA sequence dna to Human Oral Cavity microorganism group sign over time.
sample nucleic
In 16 hours after rinsing one's teeth with water, with the interval of 1 hour, according to the specification sheets of manufacturers, use OMNIgene-DISCOVER sample collecting test kit (DNAGenotek) separate microorganism genomic dna from people's saliva.Then by supersound process the DNA fragmentation of extraction changed into the mean length of 400bp and use AgencourtAMPureXP pearl (BeckmanCoulterGenomics) to carry out purifying.
be connected with the generation of the DNA fragmentation of forward adapter
The component from NuGENOvationUltralow library system (NuGENTechnologies) is utilized to produce 16 independently new-generation sequencing libraries from the sample of 100ng purifying.In brief, make DNA flat end, and prepare to connect under the Typical end described in test kit repairs reaction conditions.Fragment is only connected to forward adapter subsequently.As shown in Figure 2, connect and each end of forward adapter and each DNA fragmentation is linked together, thus produce single-stranded nick on opposite strand.Carry out adapter to fill, produce when therefore there is spacer end on each Insert Fragment and connect product.
At least the connection product of 100bp length is by carrying out purifying with the selective binding of AgencourtAMPureXP pearl, and carries out enrichment process subsequently.
primer extension
Produce by introducing reverse adapter the library containing ribosomal gene, this reverse adapter is connected to the 5 ' end these intragenic conservative regions to specific oligonucleotide.There are two different steps: the 1) annealing of gene-specific primer; And 2) by the effect of archaeal dna polymerase to the extension of this primer.The product obtained is the functional library comprising forward adapter and comprise reverse adapter on an end on another end.Use comprises 3 ' gene specific region and extends step containing the oligonucleotide in 5 ' region of the reverse adapter sequence of some Illumina to complete gene-specific primer.The variable region with 8 bases is embedded in reverse adapter sequence, and this adapter and 16 kinds of other adapters being used for other samples make a distinction by this variable region.Therefore, 16 gene specific libraries have been created; Each sample produces a library.Each library has common forward adapter.Consensus is also contained in each library in opposite ends, but in this consensus, have 8 unique nucleotide region.Compare the rrna operon from 40 kinds of different bacterium kinds by use ClustalW Multiple sequence alignments program (European Bioinformatics institute), select the total 16S sequence forming gene specific section.Synthesize following oligonucleotide and mix with equimolar ratio example, this oligonucleotide represents each in the block of 18 high conservatives identified in 16S genomic gene seat.
In 16 independently reaction, the single sample and primer extension probes (as mentioned above) with the forward adapter be connected on every bar chain are combined.They mix with the main mixture (QIAGEN, USA) of HotStarTaqPCR containing damping fluid, dNTP and heat activated Taq DNA polymerase.This solution is placed in thermal cycler, be heated to 95 DEG C keep 15 minutes to activate polysaccharase, and be cooled to 70 DEG C maintenances 5 minutes to make 16S primer and DNA Insert Fragment anneal and to extend to forward adapter site.
amplification
Merged by 16 independent primer extension products (as above), add amplimer, this amplimer is not only complementary to the 5 ' end in forward and reverse adapter site but also comprises the part with the complementation of flow cell oligonucleotide sequence.By using 3 step temperature course, (94 DEG C keep 30 seconds, 60 DEG C keep 30 seconds, and 72 DEG C keep 1 minute) PCR that carries out 25 circulations carried out to the fragment containing forward and reverse (5 ' common region on 16S primer) adapter and respective general priming site selection.Use AMPureXP pearl purified pcr product and analyze with 2100 biological analysers (AgilentTechnologies).
order-checking and data analysis
Use MiSeq system (Illumina), the test library for contrast and enrichment obtains single end sequencing reading that length is 100nt.Software processes raw sequencing data to use Illumina base to judge.Sample from different time points is put in storage (bin) and maps to ribosomal RNA database by the 8 base codes based on their uniquenesses.By the sequence mapping that do not line up with bacterium rRNA to the mankind and bacterium full-length genome reference sequences.The change of microbial population is assessed by the 16S reading counting from different organism in comparative sample in time.
The sign of the transcriptional activity of the intragroup individual cells of embodiment 3-.
sample nucleic
FACS cell sorter is used to be separated individual cells from whole blood.By cell suspension in the Prelude cracked solution (component of NuGENTechnologies, OneDirect system) of 10 μ l, cause cell membrane lysis and nuclear membrane keeps complete.16 single-cell suspension liquid are selected to be used for expression pattern analysis.In brief, as described in manufacturers, use kit reagent, produce the first and second chain cDNA by the total serum IgE existed in lysate.Use AgencourtAMPureXP pearl (BeckmanCoulterGenomics) purifying double-strand cDNA product.
be connected with the generation of the fragment of forward adapter
Use the component from NuGENOvationUltralow library system (NuGENTechnologies), produce new-generation sequencing library by the sample of each purifying.In brief, make DNA flat end, and prepare to connect under the Typical end described in test kit repairs reaction conditions.Fragment is only connected to forward adapter subsequently.As shown in Figure 2, connect and each end of forward adapter and each DNA fragmentation is linked together, thus produce single-stranded nick on opposite strand.Carry out adapter to fill, produce when therefore there is spacer end on each Insert Fragment and connect product.
At least the connection product of 100bp length is by carrying out purifying with the selective binding of AgencourtAMPureXP pearl, and carries out enrichment process subsequently.
primer extension
Library is produced by introducing the reverse adapter being connected to 5 ' end of random hexamer.There are two different steps: the 1) annealing of primer; And 2) by the effect of archaeal dna polymerase to the extension of this primer.The product obtained is the functional library comprising forward adapter and comprise reverse adapter on an end on another end.Use comprises 3 ' random areas and completes primer extension procedures containing the oligonucleotide in 5 ' region of the reverse adapter sequence of some Illumina.The variable region with 8 bases is embedded in reverse adapter sequence, and this adapter and 16 kinds of other adapters being used for other samples make a distinction by this variable region.Therefore, 16 libraries have been created; Each sample produces a library.Each library has common forward adapter.Consensus is also contained in each library in opposite ends, but in this consensus, have 8 unique nucleotide region.
In 16 independent reactions, the independent sample and primer extension probes (as mentioned above) with the forward adapter be connected on every bar chain are combined.They mix with the main mixture (QIAGEN, USA) of HotStarTaqPCR containing damping fluid, dNTP and heat activated Taq DNA polymerase.This solution is placed in thermal cycler, be heated to 95 DEG C keep 15 minutes to activate polysaccharase, and be cooled to 70 DEG C maintenances 5 minutes to make primer and DNA Insert Fragment anneal and to extend to forward adapter site.
amplification
Amplimer is added into 16 independent primer extension products (as mentioned above), this amplimer is not only complementary to the 5 ' end in forward and reverse adapter site but also comprises the part with the complementation of flow cell oligonucleotide sequence.The selection to the fragment containing forward and reverse adapter and respective general priming site has been carried out by the PCR using 3 step temperature course (94 DEG C keep 30 seconds, and 60 DEG C keep 30 seconds, and 72 DEG C keep 1 minute) to carry out 25 circulations.Use AMPureXP pearl purified pcr product and analyze with 2100 biological analysers (AgilentTechnologies).
order-checking and data analysis
Each amplification library (as mentioned above) of equal in quality is merged, and is diluted to working concentration according to manufacturer's recommendation.Use MiSeq system (Illumina), obtain for library single end sequencing reading that length is 100nt.Software processes raw sequencing data to use Illumina base to judge.Based on the 8 base codes of their uniquenesses the sample from different time points put in storage and map to reference database.Based on map feature, independent sample or new sample pool can rerun to obtain the larger reading degree of depth on sequenator.Gene covers poor sample and will remove from this pond.

Claims (78)

1., for a method for enrich target nucleotide sequence in the sample comprising nucleic acid, the method comprises:
A. the nucleic acid in sample described in fragmentation, thus produce nucleic acid fragment, wherein said nucleic acid fragment comprises target nucleic acid sequence;
B. the first adapter is connected to each 5 ' end in described nucleic acid fragment, produces the nucleic acid fragment of the first adapter sequence of 5 ' the end connection being included in described nucleic acid fragment thus;
C. the one or more oligonucleotide in solution is made to anneal with the target nucleic acid sequence in the described nucleic acid fragment comprising the first adapter sequence be connected, each in wherein said one or more oligonucleotide comprises the 3 ' part with at least 10 bases of described target nucleic acid sequence complementation, with the 5 ' part comprising the second adapter sequence, wherein said second adapter sequence has tail adapter sequence, its can not be attached to described in comprise other adapter sequence hybridizations of the nucleic acid fragment of the first adapter sequence be connected;
D. in the reactive mixture with one or more oligonucleotide that polymerase extension is annealed with the target nucleic acid sequence in the described nucleic acid fragment comprising the first adapter sequence be connected, wherein said reaction mixture be not included in extension process with the primer of sequence anneals being complementary to described first adapter sequence, thus produce one or more oligonucleotide extension products, described oligonucleotide extension products be included in the first end with the sequence of described the first adapter complementation be connected, with the sequence of described target nucleic acid sequence complementation, with the second adapter sequence in the second end, and
E. one or more oligonucleotide extension products described in the second primer amplification using first primer of annealing with the complementary sequence of described first adapter and anneal with the complementary sequence of described second adapter sequence, with target nucleic acid sequence described in enrichment, wherein said first adapter, described second adapter, described first primer, or described second primer is included in the sequence of initial order-checking on extensive parallel order-checking platform.
2. method according to claim 1, comprises the additional step checked order to the target nucleic acid sequence of described enrichment further.
3. method according to claim 2, wherein said order-checking comprises bridge amplification.
4. method according to claim 2, wherein said order-checking comprises the reversible terminator of use four kinds mark.
5. method according to claim 2, wherein said order-checking comprises nanoporous order-checking.
6. method according to claim 2, wherein said order-checking comprises semi-conductor order-checking.
7. method according to claim 1, wherein said target nucleic acid sequence comprises cDNA, RNA or from genomic DNA.
8. method according to claim 1, wherein said target nucleic acid sequence comprises from genomic DNA.
9. method according to claim 1, wherein said target nucleic acid sequence comprises cDNA.
10. method according to claim 1, each in wherein said one or more oligonucleotide comprises the 3 ' part from the different target nucleic acid sequence complementation in described nucleic acid fragment, and comprises 5 ' part of described second adapter sequence.
11. methods according to claim 1, wherein said first adapter is common for each nucleic acid fragment.
12. methods according to claim 1, wherein said second adapter sequence is common for described one or more oligonucleotide.
13. methods according to claim 1, wherein said first adapter and described second adapter sequence different from each other.
14. methods according to claim 1, wherein said first adapter and/or described second adapter sequence comprise bar code sequence.
15. methods according to claim 1, wherein said nucleic acid fragment is double-strand.
16. methods according to claim 15, make described nucleic acid fragment sex change before being included in step c further, thus produce the single-chain nucleic acid fragment at 5 ' end with described first adapter sequence.
17. methods according to claim 1, wherein said first adapter sequence comprises restriction for nucleic acid modifying enzyme and/or cleavage site.
18. methods according to claim 1, each 3 ' part in wherein said one or more oligonucleotide comprises stochastic sequence.
19. methods according to claim 1, wherein said polysaccharase is archaeal dna polymerase.
20. methods according to claim 1, are included in further after described first adapter connects and carry out breach reparation to produce the additional step with the nucleic acid fragment of spacer end.
21. methods according to claim 1, wherein said first adapter sequence or described second adapter sequence are included in the sequence of initial order-checking on extensive parallel order-checking platform, the sequence of wherein said initial order-checking on extensive parallel order-checking platform and sequencing primer complementation.
22. methods according to claim 1, wherein said first adapter sequence or described second adapter sequence are included in the sequence of initial order-checking on extensive parallel order-checking platform, and the sequence of wherein said initial order-checking on extensive parallel order-checking platform is sequencing primer sequence.
23. methods according to claim 1, the sequence of wherein said initial order-checking on extensive parallel order-checking platform is flow cell sequence.
24. methods according to claim 1, wherein said first adapter or described second adapter comprise the sequence of described initial order-checking on extensive parallel order-checking platform.
25. methods according to claim 1, wherein said first adapter and described second adapter comprise the sequence of described initial order-checking on extensive parallel order-checking platform.
26. methods according to claim 1, wherein said first primer or described second primer comprise the sequence of described initial order-checking on extensive parallel order-checking platform.
27. methods according to claim 1, wherein said first primer and described second primer comprise the sequence of described initial order-checking on extensive parallel order-checking platform.
28. methods according to claim 1, the sequence of wherein said initial order-checking on extensive parallel order-checking platform allows the target nucleic acid sequence of described enrichment and solid surface to anneal.
29. methods according to claim 28, wherein said solid surface is configured to for described extensive parallel order-checking platform.
30. methods according to claim 28, wherein said solid surface is pearl.
31. 1 kinds of methods for enrich target nucleotide sequence in the sample comprising nucleic acid, the method comprises:
A. the nucleic acid in sample described in fragmentation, thus produce nucleic acid fragment, wherein said nucleic acid fragment comprises described target nucleic acid sequence;
B. the first adapter is connected to described nucleic acid fragment, wherein said first adapter comprises the partial duplex with short chain and long-chain, 3 ' end of the short chain of wherein said partial duplex adapter comprises blocking groups, and 5 ' end of the long-chain of described partial duplex adapter comprises restriction for nucleic acid modifying enzyme and/or cleavage site;
C. make described nucleic acid fragment sex change, thus produce single-chain nucleic acid fragment, it is included in the long-chain of described first adapter on 5 ' end, described target nucleic acid sequence, and the short chain of described first adapter on 3 ' end;
D. make the target nucleic acid sequence in one or more oligonucleotide and described single-chain nucleic acid fragment anneal, each in wherein said one or more oligonucleotide comprises the sequence with the target nucleic acid sequence complementation be present in one or more described single-chain nucleic acid fragment;
E. with oligonucleotide one or more described in polymerase extension to produce one or more double-strandednucleic acid mixture, described mixture comprises described target nucleic acid sequence and complementary sequence thereof, have for the double-strand restriction of described nucleic acid modifying enzyme and/or the first end of cleavage site and second end of 3 ' overhang with the described short chain comprising described first adapter;
F. cut the restriction of described double-strand and/or cleavage site with described nucleic acid modifying enzyme, thus produce cleavage site;
G. the second adapter is connected to described cleavage site, wherein said second adapter comprises the duplex with two chains;
H. make the sex change of described one or more double-strandednucleic acid mixture, thus produce the short chain of one or more the single-chain nucleic acid fragment, the chain from described second adapter in the first end and described first adapter in the second end that comprise described target nucleic acid sequence; And
I. use and one or more single-chain nucleic acid fragment described in first primer of annealing from the complementary sequence of the chain of described second adapter and second primer amplification of annealing with the short chain of described first adapter, described single-chain nucleic acid fragment comprises described target nucleic acid sequence, at the chain from described second adapter of the first end, with the short chain of described first adapter in the second end, thus target nucleic acid sequence described in enrichment.
32. methods according to claim 31, to the additional step that the described one or more single-chain nucleic acid fragments from step I check order after being included in amplification further.
33. methods according to claim 31, wherein said target nucleic acid sequence comprises cDNA, RNA or from genomic DNA.
34. methods according to claim 31, wherein said target nucleic acid sequence comprises from genomic DNA.
35. methods according to claim 31, wherein said target nucleic acid sequence comprises cDNA.
36. methods according to claim 31, wherein said first adapter and described second adapter are common for each described nucleic acid fragment.
37. methods according to claim 31, wherein said first adapter and described second adapter different from each other.
38. methods according to claim 31, wherein said first adapter and/or described second adapter comprise bar code sequence.
39. methods according to claim 31, wherein from described in step f for the double-strand restriction of nucleic acid modifying enzyme and/or cleavage site comprise the partial duplex long-chain of described first adapter 5 ' end and is produced by the extension of described one or more oligonucleotide, and the sequence of 5 ' termini-complementary of partial duplex long-chain of described first adapter.
40. methods according to claim 31, wherein said nucleic acid modifying enzyme comprises restriction enzyme.
41. methods according to claim 31, wherein step b is undertaken by connection.
42. methods according to claim 31, wherein said polysaccharase is archaeal dna polymerase.
43. 1 kinds of methods for generation of target nucleic acid sequence library, the method comprises:
A. fragmentation comprises the sample of nucleic acid, thus produces nucleic acid fragment;
B. the first adapter is connected to each described nucleic acid fragment;
C. make described nucleic acid fragment sex change, thus produce the library of single-chain nucleic acid fragment;
D. one or more oligonucleotide and described single-chain nucleic acid fragment is made to anneal, each in wherein said one or more oligonucleotide comprises and 3 ' part of at least 10 bases of the target nucleic acid sequence complementation in one or more single-chain nucleic acid fragment and the 5 ' part comprising the second adapter sequence, wherein said second adapter sequence has tail adapter sequence, its can not be attached to described in comprise other adapter sequence hybridizations of the nucleic acid fragment of the first adapter sequence be connected;
E. one or more oligonucleotide described in reaction mixture polymerase extension, wherein said reaction mixture be not included in extension process with the primer of sequence anneals being complementary to described first adapter sequence ,thus produce one or more oligonucleotide extension products, sequence that is that described oligonucleotide extension products is included in the first end and described first adapter complementation that is that be connected, with the sequence of described target nucleic acid sequence complementation, and the described second adapter sequence in the second end; And
F. one or more oligonucleotide extension products described in the second primer amplification using first primer of annealing with the complementary sequence of described first adapter and anneal with the complementary sequence of described second adapter, to produce the library of the nucleic acid fragment comprising described first adapter and described second adapter sequence in each end, wherein said first adapter, described second adapter, described first primer, or described second primer is included in the sequence of initial order-checking on extensive parallel order-checking platform.
44. methods according to claim 43, are included in further after the first adapter connects and carry out breach reparation reaction to produce the additional step with the nucleic acid fragment of spacer end.
45. methods according to claim 43, comprise the additional step checked order to one or more oligonucleotide extension products of the amplification from step f further.
46. methods according to claim 43, wherein said order-checking comprises bridge amplification.
47. methods according to claim 43, wherein said order-checking comprises the reversible terminator of use four kinds mark.
48. methods according to claim 43, wherein said order-checking comprises nanoporous order-checking.
49. methods according to claim 43, wherein said order-checking comprises semi-conductor order-checking.
50. methods according to claim 43, wherein said target nucleic acid sequence comprises from genomic DNA.
51. methods according to claim 43, wherein said target nucleic acid sequence comprises cDNA.
52. methods according to claim 43, wherein 3 ' part of described one or more oligonucleotide of steps d comprises stochastic sequence.
53. methods according to claim 43, wherein said first adapter and described second adapter sequence are common for each nucleic acid fragment.
54. methods according to claim 43, wherein said first adapter and described second adapter sequence different from each other.
55. methods according to claim 43, wherein said first adapter and/or described second adapter sequence comprise bar code sequence.
56. methods according to claim 43, wherein said polysaccharase is archaeal dna polymerase.
57. methods according to claim 43, wherein said first adapter sequence or described second adapter sequence are included in the sequence of initial order-checking on extensive parallel order-checking platform, the sequence of wherein said initial order-checking on extensive parallel order-checking platform and sequencing primer complementation.
58. methods according to claim 43, wherein said first adapter sequence or described second adapter sequence are included in the sequence of initial order-checking on extensive parallel order-checking platform, and the sequence of wherein said initial order-checking on extensive parallel order-checking platform is sequencing primer sequence.
59. methods according to claim 43, the sequence of wherein said initial order-checking on extensive parallel order-checking platform is flow cell sequence.
60. methods according to claim 43, wherein said first adapter sequence or described second adapter sequence are included in the sequence of initial order-checking on described extensive parallel order-checking platform.
61. methods according to claim 43, wherein said first adapter sequence and described second adapter sequence are included in the sequence of initial order-checking on described extensive parallel order-checking platform.
62. methods according to claim 43, wherein said first primer or described second primer are included in the sequence of initial order-checking on described extensive parallel order-checking platform.
63. methods according to claim 43, wherein said first primer and described second primer are included in the sequence of initial order-checking on described extensive parallel order-checking platform.
64. methods according to claim 43, the sequence of wherein said initial order-checking on extensive parallel order-checking platform allows the target nucleic acid sequence of enrichment and solid surface to anneal.
65. methods according to claim 64, wherein said solid surface is configured to for described extensive parallel order-checking platform.
66. methods according to claim 64, wherein said solid surface is pearl.
67. 1 kinds of methods from library enrich target nucleotide sequence, wherein said library comprises Nucleic acid inserts, described Nucleic acid inserts comprises described target nucleic acid sequence, wherein said Nucleic acid inserts has the first adapter sequence of attachment and on the second end, has the second adapter sequence of attachment on the first end, and the method comprises:
A. make the described Nucleic acid inserts sex change that there is described first adapter sequence on the first end and there is described second adapter sequence on the second end, thus produce the library of the single-chain nucleic acid Insert Fragment that there is on the first end described first adapter sequence and there is described second adapter sequence on the second end;
B. make one or more oligonucleotide and there is on the first end described first adapter sequence and the described target nucleic acid sequence had on the second end in the described single-chain nucleic acid Insert Fragment of described second adapter sequence is annealed, each in wherein said one or more oligonucleotide comprises and 3 ' part of the described target nucleic acid sequence complementation be present in described Nucleic acid inserts and the 5 ' part comprising the 3rd adapter sequence, and wherein said 3rd adapter sequence is different with described second adapter sequence from described first adapter sequence;
C. described one or more oligonucleotide described in polymerase extension is used, thus produce one or more oligonucleotide extension products, described oligonucleotide extension products be included in the first end with the sequence of described first adapter complementary, with the sequence of described target nucleic acid sequence complementation, and the described 3rd adapter sequence in the second end; And
D. one or more oligonucleotide extension products described in the second primer amplification using first primer of annealing with the complementary sequence of described first adapter sequence and anneal with the complementary sequence of described 3rd adapter sequence, thus target nucleic acid sequence described in enrichment.
68. methods according to claim 67, comprise further and checking order to the target nucleic acid sequence of described enrichment.
69. methods according to claim 67, wherein said target nucleic acid sequence comprises from genomic DNA.
70. methods according to claim 67, wherein said target nucleic acid sequence comprises cDNA.
71. methods according to claim 67, wherein said first adapter sequence and described second adapter sequence are common for each Nucleic acid inserts in library.
72. methods according to claim 67, wherein said 3rd adapter sequence is common for described one or more oligonucleotide.
73. methods according to claim 67, wherein said first adapter sequence and described second adapter sequence different from each other.
74. methods according to claim 67, wherein said first adapter sequence and described second adapter sequence are identical.
75. methods according to claim 67, wherein said first adapter sequence, described second adapter sequence and/or described 3rd adapter sequence comprise bar code sequence.
76. methods according to claim 67, each 3 ' part in wherein said one or more oligonucleotide comprises stochastic sequence.
77. methods according to claim 1, each 3 ' part in wherein said one or more oligonucleotide is designed to hybridize with described target nucleic acid sequence.
78. according to the method for claim 43, and each 3 ' part in wherein said one or more oligonucleotide is designed to hybridize with described target nucleic acid sequence.
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